Compositions and methods for detecting human papillomavirus nucleic acid

ABSTRACT

Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of a human papillomavirus (HPV) nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/441,815, filed May 8, 2015 and now allowed, which is a 371 ofPCT/US2013/064519, filed Oct. 11, 2013, which claims benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/712,332filed Oct. 11, 2012, the entire contents of each are incorporated hereinby reference.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Dec. 7, 2017, isnamed “GP293-03-CN1_ST25.txt” and is 35 KB in size.

BACKGROUND

131 Human papillomaviruses (HPV) target epithelial tissues for infectionand are etiological agents of a variety of cancers, predominantlysquamous cell carcinomas and adenocarcinomas. HPV-associated cancersinclude those of the head and neck (larynx, oral cavity, oropharynx,tonsils, and esophagus), respiratory tissue, breast, skin, cervix, andanus. Although HPV infection is considered a necessary factor indevelopment of some cancers, other factors may also affectcarcinogenesis (Braakhuis et al., J. Natl. Cancer Inst. 96:998-1006,2004; Dahlstrand et al., Anticancer Res. 24:1829-35, 2004; Dating etal., Cancer 101:270-80, 2004; Ha et al., Crit. Rev. Oral Biol. Med.15:188-96, 2004; Hafkamp et al., Acta Otolaryngol. 124:520-6, 2004;Harwood et al., Br. J. Dermatol. 150:949-57, 2004; Rees et al., Clin.Otolaryngol. 29:301-6, 2004; Widschwendter et al., J. Clin. Virol.31:292-7, 2004).

At least 77 different types of HPV have been identified. Of those, HPV16and HPV18 are frequently linked to a variety of HPV-associated cancers,but the risk level associated with a HPV type may vary with differentforms of papilloma-associated cancers. The pathogenesis of humanpapillomaviruses in epithelia has been studied to elucidate the link ofHPV infection to cancers. HPV infects basal layer cells of stratifiedepithelia where they become established as multicopy episomes orintegrated genomes, by which the viral DNA is replicated with cellularchromosomes (reviewed by Longworth et al., Microbiol. Mol. Biol. Rev.68:362-72, 2004). At cell division, a daughter cell migrates away fromthe basal layer and undergoes differentiation in which HPV vegetativeviral replication and late-gene expression are activated to produceprogeny HPV. Although an infected individual's immune system may clearthe HPV infection, usually within 1 to 2 years, infected basal cells maypersist for decades. HPV infection may lead to chromosomal instabilityand aneuploidy that may favor HPV integration (Melsheimer et al., Clin.Cancer Res. 10:3059-63, 2004; Reidy et al., Laryngoscope 114:1906-9,2004). During HPV genome integration, the HPV E2 gene may be destroyed,resulting in deregulated expression of the HPV E6/E7 oncogenes thatencode oncoproteins that target the regulatory proteins pRb and p53.Thus, a cascade of events that modulate cellular regulation may resultin carcinogenesis (Braun et al., Cancer Lett. 209:37-49, 2004; Fan etal., Crit. Rev. Eukaryot. Gene Expr. 14:183-202, 2004; Fiedler et al.,FASEB J. 18:1120-2, 2004; Psyrri et al., Cancer Res., 64:3079-86, 2004;Si et al., J. Clin. Virol. 32:19-23, 2004).

The association of HPV infection and cervical cancer has been thesubject of considerable research and epidemiological study because ofthe high incidence of cervical cancer worldwide, estimated at 450,000new cases per year. HPV types associated with a high risk of developingcervical cancer (HR-HPV) include HPV types 16, 18, 31, 33, 35, 39, 45,51, 52, 56, 58, 59, 66, 68, and 73, although the epidemiologicalsignificance of individual types may vary with different geographicalregions or clinical testing parameters (Munoz et al., Int. J. Cancer111:278-85, 2004; Chaturvedi et al., J. Med. Virol. 75:105-13, 2005;Smith et al., Int. J. Gynaecol. Obstet. 87:131-7, 2004). HPV infectionsthat are generally considered a low risk for developing into cervicalcancer (LR-HPV) include HPV types 6, 11, 43, 43, 44, 61, 71, and 72.

In women infected with HPV, cervical infection may lead to condylomata(genital warts), cervical intraepithelial neoplasia (CIN), and cervicalcancer (Kahn et al., Adolesc. Med. Clin. 15:301-21, ix, 2004).Cytological examination of cervical cells has been the primary screeningtool for detecting cervical cancer in many countries, usually using theCIN grading system (1 to 3) to monitor precancerous lesions fordetermining treatment and/or further monitoring. In addition tocytological screening, molecular screening for HPV nucleic acid may be acost-effective prognostic test that may allow extending the timeinterval between cytological tests (Wiley et al., Curr. Oncol. Rep.6:497-506, 2004; Zielinski et al., Obstet. Gynecol. Surv. 59:543-53,2004; Clavel et al., Br. J. Cancer 90:1803-8, 2004). Molecular assayshave been developed for detection of selected HPV proteins and nucleicacid sequences in human biological specimens, e.g., Pap smears andbiopsies (Chen et al., Gynecol. Oncol. 99:578-84, 2005; Carozzi et al.,Am. J. Clin. Pathol. 124:716-21, 2005; Molden et al., Cancer Epidemiol.Biomarkers Prev. 14:367-72, 2005; Asato et al., J. Infect. Dis.189:1829-32, 2004; Federschneider et al., Am. J. Obstet. Gynecol.191:757-61, 2004; Remmerbach et al., J. Clin. Virol. 30:302-8, 2004).

Vaccination against common HPV types may be useful to treat or preventgenital warts, or prevent development of cancers, particularly cervicalcancers. Various forms of HPV vaccinations are available or are indevelopment (Ault et al., Vaccine 22:3004-7, 2004; Corona Gutierrez etal., Hum. Genet. Ther. 15:421-31, 2004; Harper et al., Lancet364:1757-65, 2004; Roden et al., Hum. Pathol. 35:971-82, 2004).

There is a need to efficiently and sensitively detect the presence ofHPV in biological specimens to provide diagnostic and prognosticinformation to physicians treating patients infected with HPV,particularly for women whose cervical tissue has been infected withHR-HPV types. There is also a need to efficiently and sensitively detectthe presence of HPV in biological specimens obtained from individualswho have been vaccinated against HPV infection, to determine theshort-term and long-term efficacy of the vaccination.

SUMMARY

In one aspect, the present invention provides a combination of at leasttwo oligomers for detecting a human papillomavirus type 33 (HPV33)target nucleic acid in a sample suspected of containing HPV33.Typically, the oligomer combination includes first and secondamplification oligomers for specifically amplifying an HPV33 nucleicacid target region corresponding to the HPV33 E6 and/or E7 gene(s). Incertain embodiments, (a) the first HPV33 amplification oligomercomprises a first target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides contained in the sequence of SEQ IDNO:66 and that includes at least the sequence of SEQ ID NO:67, SEQ IDNO:68, or SEQ ID NO:69; and (b) the second HPV33 amplification oligomercomprises a second target-hybridizing sequence that is from about 15 toabout 27 contiguous nucleotides contained in the sequence of SEQ IDNO:70 and that includes at least the sequence of SEQ ID NO:71, SEQ IDNO:72, or SEQ ID NO:73. Particularly suitable first and second HPV33amplification oligomers include (a) a first HPV33 amplification oligomercomprising a first target-hybridizing sequence selected from SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, and SEQ ID NO:8; and (b) a second HPV33 amplificationoligomer comprising a second target-hybridizing sequence selected fromSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In typical variations, theoligomer combination is for detecting an HPV33 target nucleic acid in asample suspected of containing HPV33 and at least one other HPV genotype(e.g., at least one of HPV types 16, 18, 31, 45, 52, and 58). In somevariations the oligomer combination for detecting HPV 33 and at leastone other HPV genotype includes a combination of oligomers substantiallyidentical to two or more oligomers in Table 4.

In some preferred variations, the oligomer combination further includesfirst and second amplification oligomers for specifically amplifying anHPV type 31 (HPV31) nucleic acid target region corresponding to theHPV31 E6 and/or E7 gene(s). In some such embodiments, the first HPV31amplification oligomer comprises a first target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides contained inthe sequence of SEQ ID NO:74 and that includes at least the sequence ofSEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77; and the second HPV31amplification oligomer comprises a second target-hybridizing sequencethat is from about 15 to about 30 contiguous nucleotides contained inthe sequence of SEQ ID NO:78 and that includes at least the sequence ofSEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:81. Particularly suitable firstand second HPV31 amplification oligomers include (a) a first HPV31amplification oligomer comprising a first target-hybridizing sequenceselected from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; and (b) asecond HPV33 amplification oligomer comprising a secondtarget-hybridizing sequence selected from SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40,and SEQ ID NO:41.

In some variations, the second HPV33 amplification oligomer is apromoter primer or promoter provider further comprising a promotersequence located 5′ to the target-hybridizing sequence. Similarly, insome embodiments further comprising HPV31 amplification oligomers, thesecond HPV31 amplification oligomer is a promoter primer or promoterprovider further comprising a promoter sequence located 5′ to thetarget-hybridizing sequence. Suitable promoter sequences include T7 RNApolymerase promoter sequences such as, e.g., the sequence shown in SEQID NO:82. In more specific embodiments, the second HPV33 amplificationoligomer has the sequence shown in SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ IDNO:24; and/or the second HPV31 amplification oligomer has the sequenceshown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49.

An oligomer combination may also include at least one capture probeoligomer. For example, the oligomer combination may further include anHPV33-specific capture probe oligomer and/or an HPV31-specific captureprobe oligomer. Such HPV33- or HPV31-specific capture probe oligomerstypically include a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. Suitable HPV33and HPV31 target-hybridizing sequences are shown in SEQ ID NO:50 and SEQID NO:52, respectively. In particular variations, the HPV33 or HPV31capture probe oligomer has a sequence as shown in SEQ ID NO:51 or SEQ IDNO:53, respectively.

In some embodiments, an oligomer combination further includes at leastone HPV-33 specific and/or HPV31-specific detection probe oligomer.Suitable HPV33 detection probes include oligomers comprising atarget-hybridizing sequence that is from about 14 to about 35nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:83 from about nucleotideposition 128 to about nucleotide position 164 (e.g., atarget-hybridizing sequence selected from SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58). Suitable HPV31 detectionprobes include oligomers comprising a target-hybridizing sequence thatis from about 14 to about 40 nucleotides in length and configured tospecifically hybridize to a target sequence contained within SEQ IDNO:84 from about nucleotide position 675 to about nucleotide position735 (e.g., a target-hybridizing sequence selected from SEQ ID NO:59, SEQID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, andSEQ ID NO:65).

In other aspects, the present invention provides a kit or a reactionmixture comprising an oligomer combination as above.

In yet another aspect, the present invention provides a method fordetecting, in a sample, a human papillomavirus type 33 (HPV33) targetnucleic acid (e.g., an HPV33 E6/E7 mRNA transcript). The methodgenerally includes the following steps: (a) providing a sample suspectedof containing HPV33; (b) contacting the sample with an oligomercombination for specifically amplifying an HPV33 nucleic acid targetregion, the oligomer combination comprising (i) a first HPV33amplification oligomer comprising a first target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides contained inthe sequence of SEQ ID NO:66 and that includes at least the sequence ofSEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69, and (ii) a second HPV33amplification oligomer comprising a second target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides contained inthe sequence of SEQ ID NO:70 and that includes at least the sequence ofSEQ ID NO:71, SEQ ID NO:72, or SEQ ID NO:73; (c) performing an in vitronucleic acid amplification reaction, where any HPV33 target nucleic acidpresent in said sample is used as a template for generating an HPV33amplification product; and (d) detecting the presence or absence of theHPV33 amplification product, thereby indicating the presence or absenceof HPV33 in the sample. Particularly suitable first and second type 33amplification oligomers include (a) a first HPV33 amplification oligomercomprising a first target-hybridizing sequence selected from SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, and SEQ ID NO:8; and (b) a second HPV33 amplificationoligomer comprising a second target-hybridizing sequence selected fromSEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In typical variations of themethod, the oligomer combination is for detecting an HPV33 targetnucleic acid in a sample suspected of containing HPV33 and at least oneother HPV genotype (e.g., at least one of HPV types 16, 18, 31, 45, 52,and 58). In some variations of the method, the sample is further testedfor HPV 33 and at least one other HPV genotype, wherein two of more ofthe oligomers used in the method are substantially identical to those inTable 4.

In some preferred variations, the method is for further detecting in thesample an HPV type 31 (HPV31) target nucleic acid (e.g., an HPV31 E6/E7mRNA transcript). In some such variations, the method further includesthe following steps: (b′) contacting the sample with an oligomercombination for specifically amplifying an HPV31 nucleic acid targetregion, the oligomer combination comprising (i) a first HPV31amplification oligomer comprising a first target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides contained inthe sequence of SEQ ID NO:74 and that includes at least the sequence ofSEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77, and (ii) a second HPV31amplification oligomer comprising a second target-hybridizing sequencethat is from about 15 to about 30 contiguous nucleotides contained inthe sequence of SEQ ID NO:78 and that includes at least the sequence ofSEQ ID NO:79, SEQ ID NO:80, or SEQ ID NO:81; (c′) performing an in vitronucleic acid amplification reaction, where any HPV31 target nucleic acidpresent in said sample is used as a template for generating an HPV31amplification product; and (d′) detecting the presence or absence of theHPV31 amplification product, thereby indicating the presence or absenceof HPV31 in the sample.

In some embodiments of a method, the second HPV33 amplification oligomeris a promoter primer or promoter provider further comprising a promotersequence located 5′ to the target-hybridizing sequence. Similarly, insome embodiments further comprising detection of an HPV31 target nucleicacid, the second HPV31 amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe target-hybridizing sequence. Suitable promoter sequences include T7RNA polymerase promoter sequences such as, e.g., the sequence shown inSEQ ID NO:82. In more specific embodiments, the second HPV33amplification oligomer has the sequence shown in SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, or SEQ ID NO:24; and/or the second HPV31 amplification oligomerhas the sequence shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49.

In certain variations of a method as above comprising detection of bothHPV33 and HPV31 target nucleic acids, amplification step (c) isperformed simultaneously with amplification step (c′) in the sameamplification reaction mixture. Typically, in such variations, detectionstep (d) is performed simultaneously with detection step (d′) in thesame detection reaction mixture.

Typically, the method for detecting the HPV33 target nucleic acidfurther includes purifying the HPV33 target nucleic acid from othercomponents in the sample before step (b). In particular embodiments, thepurifying step includes contacting the sample with at least oneHPV33-specific capture probe oligomer comprising a target-hybridizingsequence covalently attached to a sequence or moiety that binds to animmobilized probe. A particularly suitable HPV33 target-hybridizingsequence is shown in SEQ ID NO:50. In a more specific variation, anHPV33-specific capture probe oligomer has the sequence shown in SEQ IDNO:51.

In variations of the method comprising detection of an HPV31 targetnucleic acid, the method typically further includes purifying the HPV31target nucleic acid from other components in the sample before step(b′). In particular embodiments, the purifying step includes contactingthe sample with at least one HPV31-specific capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. A particularlysuitable HPV31 target-hybridizing sequence is shown in SEQ ID NO:52. Ina more specific variation, an HPV31-specific capture probe oligomer hasthe sequence shown in SEQ ID NO:53.

In some embodiments, the detection step (d) includes contacting theamplification reaction of step (c) with an HPV33 detection probeoligomer configured to specifically hybridize to the HPV33 amplificationproduct under conditions whereby the presence or absence of the HPV33amplification product is determined, thereby indicating the presence orabsence of HPV33 in the sample. In particular embodiments, the HPV33detection probe includes a target-hybridizing sequence that is fromabout 14 to about 35 nucleotides in length and is configured tospecifically hybridize to a target sequence contained within SEQ IDNO:83 from about nucleotide position 128 to about nucleotide position164. In specific variations, the HPV33 detection probetarget-hybridizing sequence is selected from SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58.

In some variations of the method comprising detection of an HPV 31target nucleic acid, the detection step (d′) includes contacting theamplification reaction of step (c) with an HPV31 detection probeoligomer configured to specifically hybridize to the HPV31 amplificationproduct under conditions whereby the presence or absence of the HPV31amplification product is determined, thereby indicating the presence orabsence of HPV31 in the sample. In particular embodiments, the HPV31detection probe includes a target-hybridizing sequence that is fromabout 14 to about 40 nucleotides in length and configured tospecifically hybridize to a target sequence contained within SEQ IDNO:84 from about nucleotide position 675 to about nucleotide position735. In specific variations, the HPV31 detection probetarget-hybridizing sequence is selected from SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ IDNO:65.

In some embodiments of a method utilizing a detection probe oligomer,the detection probe includes at least one label. In specific variations,the one or more label(s) are selected from a chemiluminescent label, afluorescent label, a quencher, or any combination thereof.

In other embodiments of a method utilizing a detection probe oligomer,for detecting the HPV33 target nucleic acid, the detection step (d)occurs during the amplification step (c); and/or for detecting the HPV31target nucleic acid, the detection (d′) occurs during the amplificationstep (c′). In some such embodiments, the detection probe comprises afluorescent label, a quencher, or both (e.g., a TaqMan detection probeor a molecular beacon).

In still other embodiments of a method utilizing a detection probeoligomer, the detection probe further comprises a non-target-hybridizingsequence. In particular embodiments, the detection probe comprising anon-target-hybridizing sequence is a hairpin detection probe such as,e.g., a molecular beacon or a molecular torch.

In certain embodiments of the method, the amplification reaction at step(c) and/or (c′) is an isothermal amplification reaction or a PCRreaction. In specific variations, the isothermal amplification reactionis a transcription-mediated amplification (TMA) reaction (e.g., areverse TMA reaction). In some embodiments of a method utilizing anisothermal or PCR amplification reaction, the reaction is a real-timeamplification reaction.

In some preferred embodiments of the method, (i) the detection step (d)includes contacting the amplification reaction of step (c) with an HPV33detection probe oligomer configured to specifically hybridize to theHPV33 amplification product under conditions whereby the presence orabsence of the HPV33 amplification product is determined, therebyindicating the presence or absence of HPV33 in the sample; (ii) thedetection step (d′) includes contacting the amplification reaction ofstep (c) with an HPV31 detection probe oligomer configured tospecifically hybridize to the HPV31 amplification product underconditions whereby the presence or absence of the HPV31 amplificationproduct is determined, thereby indicating the presence or absence ofHPV31 in the sample; (iii) step (c) is performed simultaneously withstep (c) in the same amplification reaction mixture and step (d) isperformed simultaneously with step (d′) in the same detection reactionmixture; and (iv) the HPV33 and HPV31 detection probe oligomers aredifferentially labeled. In some such embodiments, each of the HPV33 andHPV31 detection probe oligomers comprises a label independently selectedfrom a chemiluminescent label and a fluorescent label. In moreparticular variations, each of the HPV33 and HPV31 detection probeoligomers comprises a chemiluminescent label; in some such variations,the chemiluminescent labels for the HPV33 and HPV31 detection probeoligomers are characterized by different light emission kineticssufficient to distinguish between HPV33-specific and HPV31-specificchemiluminescent signals. Suitable chemiluminescent labels for the HPV33and HPV31 detection probe oligomers include labels comprising anacridinium ester (AE).

In still another aspect, the present invention provides a detectionprobe oligomer for detecting an HPV33 or HPV31 target nucleic acid. Insome embodiments, a detection probe oligomer for detecting HPV33comprises a target-hybridizing sequence that is from about 14 to about35 nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:83 from about nucleotideposition 128 to about nucleotide position 164 (e.g., atarget-hybridizing sequence selected from SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:56, SEQ ID NO:57, and SEQ ID NO:58). In some embodiments, adetection probe oligomer for detecting HPV31 comprises atarget-hybridizing sequence that is from about 14 to about 40nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:84 from about nucleotideposition 675 to about nucleotide position 735 (e.g., atarget-hybridizing sequence selected from SEQ ID NO:59, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ IDNO:65).

In some embodiments of a detection probe oligomer, the detection probeincludes at least one label. In specific variations, the one or morelabel(s) are selected from a chemiluminescent label, a fluorescentlabel, a quencher, or any combination thereof. In more specificvariations, the detection probe comprises a fluorescent label and aquencher (e.g., a TaqMan detection probe or a molecular beacon).

In other embodiments of a detection probe oligomer, the detection probefurther comprises a non-target-hybridizing sequence. In particularvariations, the detection probe comprising a non-target-hybridizingsequence is a hairpin detection probe such as, e.g., a molecular beaconor a molecular torch.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise. For example, “a nucleic acid” asused herein is understood to represent one or more nucleic acids. Assuch, the terms “a” (or “an”), “one or more,” and “at least one” can beused interchangeably herein.

“Sample” or “biological sample” refers to any tissue or material derivedfrom a living or dead human that may contain a human papillomavirus(HPV) target nucleic acid, including, e.g., samples of larynx, oralcavity, oropharynx, tonsil, or esophagus tissue, respiratory tissue orexudates, cervical or anal swab samples, biopsy tissue including lymphnodes, gastrointestinal tissue, feces, urine, semen, sputum, peripheralblood, plasma, serum or other body fluids, tissues or materials. Thebiological sample may be treated to physically or mechanically disrupttissue or cell structure, thus releasing intracellular components into asolution which may further contain enzymes, buffers, salts, detergentsand the like, which are used to prepare, using standard methods, abiological sample for analysis. Also, samples may include processedsamples, such as those obtained from passing samples over or through afiltering device, or following centrifugation, or by adherence to amedium, matrix, or support. Samples may further include cross-linkedtissues or material derived from a human, such as samples that arecontained in a media such as BD SurePath Preservative Fluid (Becton,Dickinson and Company, Franklin Lakes, N.J.), or formalin-fixedparaffin-embedded samples. Samples may further include tissues ormaterial derived from a human and are suspended in a Cytology media suchas ThinPrep Cytology Reagent (Hologic, Inc., Bedford, Mass.).

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see, e.g., International PatentApplication Pub. No. WO 95/32305), phosphorothioate linkages,methylphosphonate linkages, or combinations thereof. Sugar moieties ofthe nucleic acid may be either ribose or deoxyribose, or similarcompounds having known substitutions such as, for example, 2′-methoxysubstitutions and 2′-halide substitutions (e.g., 2′-F). Nitrogenousbases may be conventional bases (A, G, C, T, U), analogs thereof (e.g.,inosine, 5-methylisocytosine, isoguanine; see, e.g., The Biochemistry ofthe Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992; Abraham etal., 2007, BioTechniques 43: 617-24), which include derivatives ofpurine or pyrimidine bases (e.g., N⁴-methyl deoxygaunosine, deaza- oraza-purines, deaza- or aza-pyrimidines, pyrimidine bases havingsubstituent groups at the 5 or 6 position, purine bases having analtered or replacement substituent at the 2, 6 and/or 8 position, suchas 2-amino-6-methylaminopurine, O⁶-methylguanine, 4-thio-pyrimidines,4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, andO⁴-alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or3-substituted pyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825,6,949,367 and International Patent Application Pub. No. WO 93/13121,each incorporated by reference herein). Nucleic acids may include“abasic” residues in which the backbone does not include a nitrogenousbase for one or more residues (see, e.g., U.S. Pat. No. 5,585,481,incorporated by reference herein). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′-methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., Biochemistry 43:13233-41, 2004, incorporated by reference herein).Nucleic acids may include modified bases to alter the function orbehavior of the nucleic acid, e.g., addition of a 3′-terminaldideoxynucleotide to block additional nucleotides from being added tothe nucleic acid. Synthetic methods for making nucleic acids in vitroare well known in the art although nucleic acids may be purified fromnatural sources using routine techniques.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “3′-to-5′,” i.e., by the addition ofnucleotides to the 5′-terminus of a growing nucleic acid.

A “nucleotide” as used herein is a subunit of a nucleic acid consistingof a phosphate group, a 5-carbon sugar and a nitrogenous base. The5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me). As usedherein, methoxy oligonucleotides containing “T” residues have a methoxygroup at the 2′ position of the ribose moiety, and a uracil at the baseposition of the nucleotide.

A “non-nucleotide unit” as used herein is a unit that does notsignificantly participate in hybridization of a polymer. Such units mustnot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” as used herein is a nucleic acid comprising atarget sequence to be amplified. Target nucleic acids may be DNA or RNAas described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence, which may not be amplified.

By “isolated” it is meant that a sample containing a target nucleic acidis taken from its natural milieu, but the term does not connote anydegree of purification.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid that is to be amplifiedand/or detected. The “target sequence” includes the complexing sequencesto which oligonucleotides (e.g., priming oligonucleotides and/orpromoter oligonucleotides) complex during an amplification processes(e.g., TMA). Where the target nucleic acid is originallysingle-stranded, the term “target sequence” will also refer to thesequence complementary to the “target sequence” as present in the targetnucleic acid. Where the target nucleic acid is originallydouble-stranded, the term “target sequence” refers to both the sense (+)and antisense (−) strands.

“Target-hybridizing sequence” is used herein to refer to the portion ofan oligomer that is configured to hybridize with a target nucleic acidsequence. Preferably, the target-hybridizing sequences are configured tospecifically hybridize with a target nucleic acid sequence.Target-hybridizing sequences may be 100% complementary to the portion ofthe target sequence to which they are configured to hybridize, but notnecessarily. Target-hybridizing sequences may also include inserted,deleted and/or substituted nucleotide residues relative to a targetsequence. Less than 100% complementarity of a target-hybridizingsequence to a target sequence may arise, for example, when the targetnucleic acid is a plurality of strains within a species. It isunderstood that other reasons exist for configuring a target-hybridizingsequence to have less than 100% complementarity to a target nucleicacid.

Oligomer target-hybridizing sequences defined herein by reference to aspecific sequence (e.g., by reference a region within SEQ ID NO:83 orSEQ ID NO:84) are also understood to include functional complementsthereof, unless the context clearly dictates otherwise. Thus, forexample, where target-hybridizing regions of first and secondamplification oligomers are defined by reference to specific sequencescorresponding, respectively, to sense and antisense strands of a targetnucleic acid, it is understood that the amplification oligomercombination may include a functional combination of first and secondamplification oligomers having target-hybridizing sequences that are therespective complements of the specific reference sequences. Similarly,and again by way of example, where a target-hybridizing sequence for adetection probe oligomer is defined reference to a specific sequence, itis understood that the detection probe may include a correspondingdetection probe oligomer having a target-hybridizing sequence that isthe complement of the specific reference sequence; or where a detectionprobe oligomer is defined by its configuration to hybridize to aspecific sequence, it is understood that the detection probe may includea corresponding detection probe oligomer having a target-hybridizingsequence that is configured to hybridize to the complement of thespecific reference sequence.

The term “target a sequence,” as used herein in reference to a region ofan HPV nucleic acid, refers to a process whereby an oligonucleotidehybridizes to the target sequence in a manner that allows foramplification and detection as described herein. In one preferredembodiment, the oligonucleotide is complementary with the targeted HPVnucleic acid sequence and contains no mismatches. In another preferredembodiment, the oligonucleotide is complementary but contains 1, 2, 3,4, or 5 mismatches with the targeted HPV nucleic acid sequence.Preferably, the oligonucleotide that hybridizes to the HPV nucleic acidsequence includes at least 10 to as many as 50 nucleotides complementaryto the target sequence. It is understood that at least 10 and as many as50 is an inclusive range such that 10, 50 and each whole number therebetween are included. Preferably, the oligomer specifically hybridizesto the target sequence.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of a referenced oligonucleotidetarget-hybridizing sequence. For example, amplification oligomers thatare configured to generate a specified amplicon from a target sequencehave polynucleotide sequences that hybridize to the target sequence andcan be used in an amplification reaction to generate the amplicon. Alsoas an example, oligonucleotides that are configured to specificallyhybridize to a target sequence have a polynucleotide sequence thatspecifically hybridizes to the referenced sequence under stringenthybridization conditions.

The term “configured to specifically hybridize to” as used herein meansthat the target-hybridizing region of an amplification oligonucleotide,detection probe, or other oligonucleotide is designed to have apolynucleotide sequence that could target a sequence of the referencedHPV target region. Such an oligonucleotide is not limited to targetingthat sequence only, but is rather useful as a composition, in a kit orin a method for targeting an HPV target nucleic acid. Theoligonucleotide is designed to function as a component of an assay foramplification and detection of HPV from a sample, and therefore isdesigned to target HPV in the presence of other nucleic acids commonlyfound in testing samples. “Specifically hybridize to” does not meanexclusively hybridize to, as some small level of hybridization tonon-target nucleic acids may occur, as is understood in the art. Rather,“specifically hybridize to” means that the oligonucleotide is configuredto function in an assay to primarily hybridize the target so that anaccurate detection of target nucleic acid in a sample can be determined.The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of the amplificationoligonucleotide target-hybridizing sequence.

The term “fragment,” as used herein in reference to an HPV targetnucleic acid, refers to a piece of contiguous nucleic acid, wherein thenumber of contiguous nucleotides in the fragment are less than that forthe entire target nucleic acid.

The term “region,” as used herein, refers to a portion of a nucleic acidwherein said portion is smaller than the entire nucleic acid. Forexample, when the nucleic acid in reference is an oligonucleotidepromoter primer, the term “region” may be used to refer to the smallerpromoter portion of the entire oligonucleotide. Similarly, and also asexample only, when the nucleic acid is an HPV E6 and/or E7 segment of anHPV genome (e.g., an E6/E7 mRNA transcript), the term “region” may beused to refer to a smaller area of the nucleic acid, wherein the smallerarea is targeted by one or more oligonucleotides of the invention. Asanother non-limiting example, when the nucleic acid in reference is anamplicon, the term region may be used to refer to the smaller nucleotidesequence identified for hybridization by the target-hybridizing sequenceof a probe.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof about 12 to 15 nt and an upper limit of about 50 to 600 nt, and otherembodiments are in a range having a lower limit of about 15 to 20 nt andan upper limit of about 22 to 100 nt. Oligonucleotides may be purifiedfrom naturally occurring sources or may be synthesized using any of avariety of well-known enzymatic or chemical methods. The termoligonucleotide does not denote any particular function to the reagent;rather, it is used generically to cover all such reagents describedherein. An oligonucleotide may serve various different functions. Forexample, it may function as a primer if it is specific for and capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase; it may function as a primerand provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Primer); and it mayfunction to detect a target nucleic acid if it is capable of hybridizingto the target nucleic acid, or an amplicon thereof, and further providesa detectible moiety (e.g., an acridinium-ester compound).

As used herein, an oligonucleotide “substantially corresponding to” aspecified reference nucleic acid sequence means that the oligonucleotideis sufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.One skilled in the art will understand that “substantially correspondingoligonucleotides” can vary from a reference sequence and still hybridizeto the same target nucleic acid sequence. It is also understood that afirst nucleic acid corresponding to a second nucleic acid includes theRNA and DNA thereof and includes the complements thereof, unless thecontext clearly dictates otherwise. This variation from the nucleic acidmay be stated in terms of a percentage of identical bases within thesequence or the percentage of perfectly complementary bases between theprobe or primer and its target sequence. Thus, in certain embodiments,an oligonucleotide “substantially corresponds” to a reference nucleicacid sequence if these percentages of base identity or complementarityare from 100% to about 80%. In preferred embodiments, the percentage isfrom 100% to about 85%. In more preferred embodiments, this percentageis from 100% to about 90%; in other preferred embodiments, thispercentage is from 100% to about 95%. Similarly, a region of a nucleicacid or amplified nucleic acid can be referred to herein ascorresponding to a reference nucleic acid sequence. One skilled in theart will understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase. Oligomersnot intended for extension by a nucleic acid polymerase may include ablocker group that replaces the 3′ OH to prevent enzyme-mediatedextension of the oligomer in an amplification reaction. For example,blocked amplification oligomers and/or detection probes present duringamplification may not have functional 3′ OH and instead include one ormore blocking groups located at or near the 3′ end. In some embodimentsa blocking group near the 3′ end and may be within five residues of the3′ end and is sufficiently large to limit binding of a polymerase to theoligomer. In other embodiments a blocking group is covalently attachedto the 3′ terminus. Many different chemical groups may be used to blockthe 3′ end, e.g., alkyl groups, non-nucleotide linkers, alkane-dioldideoxynucleotide residues, and cordycepin.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid, and which hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a target nucleic acid and contains a 3′ OHend that is extended by a polymerase in an amplification process.Another example of an amplification oligomer is an oligomer that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. For example, the 5′ regionof an amplification oligonucleotide may include a promoter sequence thatis non-complementary to the target nucleic acid (which may be referredto as a “promoter primer” or “promoter provider”). Those skilled in theart will understand that an amplification oligomer that functions as aprimer may be modified to include a 5′ promoter sequence, and thusfunction as a promoter primer. Incorporating a 3′ blocked end furthermodifies the promoter primer, which is now capable of hybridizing to atarget nucleic acid and providing an upstream promoter sequence thatserves to initiate transcription, but does not provide a primer foroligo extension. Such a modified oligo is referred to herein as a“promoter provider” oligomer. Size ranges for amplificationoligonucleotides include those that are about 10 to about 70 nt long(not including any promoter sequence or poly-A tails) and contain atleast about 10 contiguous bases, or even at least 12 contiguous basesthat are complementary to a region of the target nucleic acid sequence(or a complementary strand thereof). The contiguous bases are at least80%, or at least 90%, or completely complementary to the target sequenceto which the amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or additionalnucleotides that participate in an amplification reaction but are notcomplementary to or contained in the target nucleic acid, or templatesequence. It is understood that when referring to ranges for the lengthof an oligonucleotide, amplicon, or other nucleic acid, that the rangeis inclusive of all whole numbers (e.g., 15-27 contiguous nucleotides inlength includes 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27).

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 50or more nucleotides in length. The “second region” comprises a promotersequence for an RNA polymerase. A promoter oligonucleotide is engineeredso that it is incapable of being extended by an RNA- or DNA-dependentDNA polymerase, e.g., reverse transcriptase, preferably comprising ablocking moiety at its 3′-terminus as described above. As referred toherein, a “T7 Provider” is a blocked promoter provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase.

A “terminating oligonucleotide” is an oligonucleotide comprising a basesequence that is substantially complementary to a sequence within thetarget nucleic acid in the vicinity of the 5′-end of the target region,so as to “terminate” primer extension of a nascent nucleic acid thatincludes a priming oligonucleotide, thereby providing a defined 3′-endfor the nascent nucleic acid strand. A terminating oligonucleotide isdesigned to hybridize to the target nucleic acid at a positionsufficient to achieve the desired 3′-end for the nascent nucleic acidstrand. The positioning of the terminating oligonucleotide is flexibledepending upon its design. A terminating oligonucleotide may be modifiedor unmodified. In certain embodiments, terminating oligonucleotides aresynthesized with at least one or more 2′-O-ME ribonucleotides. Thesemodified nucleotides have demonstrated higher thermal stability ofcomplementary duplexes. The 2′-O-ME ribonucleotides also function toincrease the resistance of oligonucleotides to exonucleases, therebyincreasing the half-life of the modified oligonucleotides. (See, e.g.,Majlessi et al., Nucleic Acids Res. 26:2224-9, 1988, incorporated byreference herein.) Other modifications as described elsewhere herein maybe utilized in addition to or in place of 2′-O-ME ribonucleotides. Forexample, a terminating oligonucleotide may comprise PNA or an LNA. (See,e.g., Petersen et al., J. Mol. Recognit. 13:44-53, 2000, incorporated byreference herein.) A terminating oligonucleotide of the presentinvention typically includes a blocking moiety at its 3′-terminus toprevent extension. A terminating oligonucleotide may also comprise aprotein or peptide joined to the oligonucleotide so as to terminatefurther extension of a nascent nucleic acid chain by a polymerase. Aterminating oligonucleotide of the present invention is typically atleast 10 bases in length, and may extend up to 15, 20, 25, 30, 35, 40,50 or more nucleotides in length. While a terminating oligonucleotidetypically or necessarily includes a 3′-blocking moiety, “3′-blocked”oligonucleotides are not necessarily terminating oligonucleotides.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, e.g., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include, for example, replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (see, e.g., U.S. Pat. No.4,786,600, incorporated by reference herein). PCR amplification uses aDNA polymerase, pairs of primers, and thermal cycling to synthesizemultiple copies of two complementary strands of dsDNA or from a cDNA(see, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,800,159; eachincorporated by reference herein). LCR amplification uses four or moredifferent oligonucleotides to amplify a target and its complementarystrand by using multiple cycles of hybridization, ligation, anddenaturation (see, e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663, eachincorporated by reference herein). SDA uses a primer that contains arecognition site for a restriction endonuclease and an endonuclease thatnicks one strand of a hemimodified DNA duplex that includes the targetsequence, whereby amplification occurs in a series of primer extensionand strand displacement steps (see, e.g., U.S. Pat. Nos. 5,422,252;5,547,861; and 5,648,211; each incorporated by reference herein).

“Transcription-associated amplification” or “transcription-mediatedamplification” (TMA) refer to nucleic acid amplification that uses anRNA polymerase to produce multiple RNA transcripts from a nucleic acidtemplate. These methods generally employ an RNA polymerase, a DNApolymerase, deoxyribonucleoside triphosphates, ribonucleosidetriphosphates, and a template complementary oligonucleotide thatincludes a promoter sequence, and optionally may include one or moreother oligonucleotides. TMA methods and single-primer transcriptionassociated amplification method are embodiments of amplification methodsused for detection of HPV target sequences as described herein.Variations of transcription-associated amplification are well known inthe art as previously disclosed in detail (see, e.g., U.S. Pat. Nos.4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516; and7,374,885; and International Patent Application Pub. Nos. WO 88/01302;WO 88/10315; and WO 95/03430; each incorporated by reference herein).The person of ordinary skill in the art will appreciate that thedisclosed compositions may be used in amplification methods based onextension of oligomer sequences by a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “amplicon” or the term “amplification product” as used hereinrefers to the nucleic acid molecule generated during an amplificationprocedure that is complementary or homologous to a sequence containedwithin the target sequence. The complementary or homologous sequence ofan amplicon is sometimes referred to herein as a “target-specificsequence.” Amplicons generated using the amplification oligomers of thecurrent invention may comprise non-target specific sequences. Ampliconscan be double stranded or single stranded and can include DNA, RNA orboth. For example, DNA-dependent RNA polymerase transcribes singlestranded amplicons from double-stranded DNA duringtranscription-mediated amplification procedures. These single-strandedamplicons are RNA amplicons and can be either strand of adouble-stranded complex, depending on how the amplification oligomersare configured. Thus, amplicons can be single-stranded RNA.RNA-dependent DNA polymerases synthesize a DNA strand that iscomplementary to an RNA template. Thus, amplicons can be double-strandedDNA and RNA hybrids. RNA-dependent DNA polymerases often include RNaseactivity, or are used in conjunction with an RNase, which degrades theRNA strand. Thus, amplicons can be single stranded DNA. RNA-dependentDNA polymerases and DNA-dependent DNA polymerases synthesizecomplementary DNA strands from DNA templates. Thus, amplicons can bedouble-stranded DNA. RNA-dependent RNA polymerases synthesize RNA froman RNA template. Thus, amplicons can be double-stranded RNA.DNA-dependent RNA polymerases synthesize RNA from double-stranded DNAtemplates, also referred to as transcription. Thus, amplicons can besingle stranded RNA. Amplicons and methods for generating amplicons areknown to those skilled in the art. For convenience herein, a singlestrand of RNA or a single strand of DNA may represent an amplicongenerated by an amplification oligomer combination of the currentinvention. Such representation is not meant to limit the amplicon to therepresentation shown. Skilled artisans in possession of the instantdisclosure will use amplification oligomers and polymerase enzymes togenerate any of the numerous types of amplicons, all within the spiritand scope of the current invention.

A “non-target-specific sequence,” as is used herein refers to a regionof an oligomer sequence, wherein said region does not stably hybridizewith a target sequence under standard hybridization conditions.Oligomers with non-target-specific sequences include, but are notlimited to, promoter primers and molecular beacons. An amplificationoligomer may contain a sequence that is not complementary to the targetor template sequence; for example, the 5′ region of a primer may includea promoter sequence that is non-complementary to the target nucleic acid(referred to as a “promoter primer”). Those skilled in the art willunderstand that an amplification oligomer that functions as a primer maybe modified to include a 5′ promoter sequence, and thus function as apromoter primer. Similarly, a promoter primer may be modified by removalof, or synthesis without, a promoter sequence and still function as aprimer. A 3′ blocked amplification oligomer may provide a promotersequence and serve as a template for polymerization (referred to as a“promoter provider”). Thus, an amplicon that is generated by anamplification oligomer member such as a promoter primer will comprise atarget-specific sequence and a non-target-specific sequence.

“Detection probe,” “detection oligonucleotide,” and “detection probeoligomer” are used interchangeably to refer to a nucleic acid oligomerthat hybridizes specifically to a target sequence in a nucleic acid, orin an amplified nucleic acid, under conditions that promotehybridization to allow detection of the target sequence or amplifiednucleic acid. Detection may either be direct (e.g., a probe hybridizeddirectly to its target sequence) or indirect (e.g., a probe linked toits target via an intermediate molecular structure). Detection probesmay be DNA, RNA, analogs thereof or combinations thereof and they may belabeled or unlabeled. Detection probes may further include alternativebackbone linkages such as, e.g., 2′-O-methyl linkages A detectionprobe's “target sequence” generally refers to a smaller nucleic acidsequence region within a larger nucleic acid sequence that hybridizesspecifically to at least a portion of a probe oligomer by standard basepairing. A detection probe may comprise target-specific sequences andother sequences that contribute to the three-dimensional conformation ofthe probe (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412;6,835,542; 6,534,274; and 6,361,945; and US Patent Application Pub. No.20060068417; each incorporated by reference herein).

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labeling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labelingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (see,e.g., U.S. Pat. Nos. 5,283,174; 5,656,207; and 5,658,737; eachincorporated by reference herein). Labels include chemiluminescentcompounds, e.g., acridinium ester (“AE”) compounds that include standardAE and derivatives (see, e.g., U.S. Pat. Nos. 5,656,207; 5,658,737; and5,639,604; each incorporated by reference herein). Synthesis and methodsof attaching labels to nucleic acids and detecting labels are wellknown. (See, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Habor,N Y, 1989), Chapter 10, incorporated by reference herein. See also U.S.Pat. Nos. 5,658,737; 5,656,207; 5,547,842; 5,283,174; and 4,581,333;each incorporated by reference herein). More than one label, and morethan one type of label, may be present on a particular probe, ordetection may use a mixture of probes in which each probe is labeledwith a compound that produces a detectable signal (see, e.g., U.S. Pat.Nos. 6,180,340 and 6,350,579, each incorporated by reference herein).

“Capture probe,” “capture oligonucleotide,” and “capture probe oligomer”are used interchangeably to refer to a nucleic acid oligomer thatspecifically hybridizes to a target sequence in a target nucleic acid bystandard base pairing and joins to a binding partner on an immobilizedprobe to capture the target nucleic acid to a support. One example of acapture oligomer includes two binding regions: a sequence-binding region(e.g., target-specific portion) and an immobilized probe-binding region,usually on the same oligomer, although the two regions may be present ontwo different oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer uses a target-sequence binding regionthat includes random or non-random poly-GU, poly-GT, or poly U sequencesto bind non-specifically to a target nucleic acid and link it to animmobilized probe on a support.

As used herein, an “immobilized oligonucleotide,” “immobilized probe,”or “immobilized nucleic acid” refers to a nucleic acid binding partnerthat joins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample. Oneembodiment of an immobilized probe is an oligomer joined to a supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Supports may include known materials, such asmatrices and particles free in solution, which may be made ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal, or other compositions, of which oneembodiment is magnetically attractable particles. Supports may bemonodisperse magnetic spheres (e.g., uniform size±5%), to which animmobilized probe is joined directly (via covalent linkage, chelation,or ionic interaction), or indirectly (via one or more linkers), wherethe linkage or interaction between the probe and support is stableduring hybridization conditions.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. Sequences that hybridize to each other may becompletely complementary or partially complementary to the intendedtarget sequence by standard nucleic acid base pairing (e.g., G:C, A:T orA:U pairing). By “sufficiently complementary” is meant a contiguoussequence that is capable of hybridizing to another sequence by hydrogenbonding between a series of complementary bases, which may becomplementary at each position in the sequence by standard base pairingor may contain one or more residues, including abasic residues, that arenot complementary. Sufficiently complementary contiguous sequencestypically are at least 80%, or at least 90%, complementary to a sequenceto which an oligomer is intended to specifically hybridize. Sequencesthat are “sufficiently complementary” allow stable hybridization of anucleic acid oligomer with its target sequence under appropriatehybridization conditions, even if the sequences are not completelycomplementary. When a contiguous sequence of nucleotides of onesingle-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “completely”complementary (see, e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51 and11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57, incorporated by reference herein). It is understood thatranges for percent identity are inclusive of all whole and partialnumbers (e.g., at least 90% includes 90, 91, 93.5, 97.687 and etc.).

By “preferentially hybridize” or “specifically hybridize” is meant thatunder stringent hybridization assay conditions, probes hybridize totheir target sequences, or replicates thereof, to form stableprobe:target hybrids, while at the same time formation of stableprobe:non-target hybrids is minimized Thus, a probe hybridizes to atarget sequence or replicate thereof to a sufficiently greater extentthan to a non-target sequence, to enable one having ordinary skill inthe art to accurately quantitate the RNA replicates or complementary DNA(cDNA) of the target sequence formed during the amplification.Appropriate hybridization conditions are well-known in the art, may bepredicted based on sequence composition, or can be determined by usingroutine testing methods (see, e.g., Sambrook et al., Molecular Cloning,A Laboratory Manual, 2^(nd) ed. (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1989) at §§ 1.90-1.91, 7.37-7.57, 9.47-9.51and 11.47-11.57, particularly §§ 9.50-9.51, 11.12-11.13, 11.45-11.47 and11.55-11.57, incorporated by reference herein).

By “nucleic acid hybrid,” “hybrid,” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

“Sample preparation” refers to any steps or method that treats a samplefor subsequent amplification and/or detection of HPV nucleic acidspresent in the sample. Samples may be complex mixtures of components ofwhich the target nucleic acid is a minority component. Samplepreparation may include any known method of concentrating components,such as microbes or nucleic acids, from a larger sample volume, such asby filtration of airborne or waterborne particles from a larger volumesample or by isolation of microbes from a sample by using standardmicrobiology methods. Sample preparation may include physical disruptionand/or chemical lysis of cellular components to release intracellularcomponents into a substantially aqueous or organic phase and removal ofdebris, such as by using filtration, centrifugation or adsorption.Sample preparation may include use of a nucleic acid oligonucleotidethat selectively or non-specifically capture a target nucleic acid andseparate it from other sample components (e.g., as described in U.S.Pat. No. 6,110,678 and International Patent Application Pub. No. WO2008/016988, each incorporated by reference herein).

“Separating” or “purifying” means that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are theDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

The term “specificity,” in the context of an amplification and/ordetection system, is used herein to refer to the characteristic of thesystem which describes its ability to distinguish between target andnon-target sequences dependent on sequence and assay conditions. Interms of nucleic acid amplification, specificity generally refers to theratio of the number of specific amplicons produced to the number ofside-products (e.g., the signal-to-noise ratio). In terms of detection,specificity generally refers to the ratio of signal produced from targetnucleic acids to signal produced from non-target nucleic acids.

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, e.g., the ratio of specific amplicons toside-products.

As used herein, the term “relative light unit” (“RLU”) is an arbitraryunit of measurement indicating the relative number of photons emitted bythe sample at a given wavelength or band of wavelengths. RLU varies withthe characteristics of the detection means used for the measurement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a reference sequence for the E6/E7 region of the HPVtype 33 genome (SEQ ID NO:83)

FIG. 2 illustrates a reference sequence for the E6/E7 region of the HPVtype 31 genome (SEQ ID NO:84).

DETAILED DESCRIPTION

The present invention provides compositions, kits and methods foramplifying and detecting human papillomavirus (HPV) nucleic acid from asample, specifically sequences corresponding to the E6/E7 region of theHPV type 33 (HPV33) and/or HPV type 31 (HPV31) genome. The compositions,kits and methods provide oligonucleotide sequences that recognize targetsequences within the HPV33 or HPV31 E6/E7 region or their complementarysequences. Such oligonucleotides may be used as amplificationoligonucleotides, which may include primers, promoter primers, blockedoligonucleotides, and promoter provider oligonucleotides, whosefunctions have been described previously (see, e.g., U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 5,399,491; 5,554,516; 5,824,518; and7,374,885; each incorporated by reference herein). Otheroligonucleotides may be used as capture probe oligomers for capturingand separating target nucleic acids from various sample components.Other oligonucleotides may be used as probes for detecting amplifiedsequences of HPV33 or HPV31.

The methods provide for the sensitive and specific detection of HPV33and/or HPV31 nucleic acids, such as, e.g., HPV33 and/or HPV31 E6/E7 mRNAtranscripts. The methods include performing a nucleic acid amplificationof HPV33 and/or HPV31 sequences and detecting the amplified product by,for example, specifically hybridizing the amplified product with anucleic acid detection probe that provides a signal to indicate thepresence of HPV33 or HPV31 in the sample. The amplification stepincludes contacting the sample with one or more amplification oligomersspecific for a target sequence in the HPV33 or HPV31 E6/E7 region toproduce an amplified product if HPV33 or HPV31 nucleic acid is presentin the sample. Amplification synthesizes additional copies of the targetsequence or its complement by using at least one nucleic acid polymeraseto extend the sequence from an amplification oligomer (a primer) using atemplate strand. One embodiment for detecting the amplified product usesa hybridizing step that includes contacting the amplified product withat least one probe specific for a sequence amplified by the selectedamplification oligomers, e.g., a sequence contained in the targetsequence flanked by a pair of selected amplification oligomers.

The detection step may be performed using any of a variety of knowntechniques to detect a signal specifically associated with the amplifiedtarget sequence, such as, e.g., by hybridizing the amplification productwith a labeled detection probe and detecting a signal resulting from thelabeled probe. The detection step may also provide additionalinformation on the amplified sequence, such as, e.g., all or a portionof its nucleic acid base sequence. Detection may be performed after theamplification reaction is completed, or may be performed simultaneouslywith amplifying the target region, e.g., in real time. In oneembodiment, the detection step allows homogeneous detection, e.g.,detection of the hybridized probe without removal of unhybridized probefrom the mixture (see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174,each incorporated by reference herein).

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. One example of such detection uses a luminescentally labeledprobe that hybridizes to target nucleic acid. Luminescent label is thenhydrolyzed from non-hybridized probe. Detection is performed bychemiluminescence using a luminometer. (see, e.g., International PatentApplication Pub. No. WO 89/002476, incorporated by reference herein). Inother embodiments that use real-time detection, the detection probe maybe a hairpin probe such as, for example, a molecular beacon, moleculartorch, or hybridization switch probe that is labeled with a reportermoiety that is detected when the probe binds to amplified product. Suchprobes may comprise target-hybridizing sequences andnon-target-hybridizing sequences. Various forms of such probes have beendescribed previously (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728;5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945;and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1;each incorporated by reference herein).

Preferred compositions of the instant invention are configured tospecifically hybridize to an E6/E7 nucleic acid target region of HPV33or HPV31 with minimal cross-reactivity to other nucleic acids suspectedof being in a sample. In some aspects, the compositions of the instantinvention are configured to specifically hybridize to an E6/E7 nucleicacid target region of HPV33 or HPV31 with minimal cross-reactivity toone or more other HPV types. For example, in certain embodiments,compositions of the invention are configured to specifically hybridizeto an E6/E7 region of HPV33 with minimal cross-reactivity to one or moreof HPV types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68; in somesuch variations, the compositions are configured to specificallyhybridize to an E6/E7 region of HPV33 with minimal cross-reactivity toat least (i) HPV31 and/or (ii) one or both of closely related HPV types52 and 58 (e.g., with minimal cross-reactivity to each of HPV types 31,52, and 58). In other embodiments, compositions of the invention areconfigure to specifically hybridize to an E6/E7 region of HPV31 withminimal cross-reactivity to one or more of HPV types 16, 18, 33, 35, 39,45, 51, 52, 56, 58, 59, and 68; in some such variations, thecompositions are configured to specifically hybridize to an E6/E7 regionof HPV31 with minimal cross-reactivity to at least (i) HPV33 and/or (ii)one or both of closely related HPV types 52 and 58 (e.g., with minimalcross-reactivity to each of HPV types 33, 52, and 58). In one aspect,the compositions of the instant invention are part of a multiplex systemthat further includes components and methods for detecting one of moreof these HPV types (e.g., a multiplex system for detecting one or moreof HPV33 and HPV31).

In certain aspects of the invention, a combination of at least twooligomers is provided for the detection of an HPV33 target nucleic acidin a sample suspected of containing HPV33. Typically, the oligomercombination includes first and second amplification oligomers forspecifically amplifying an HPV33 nucleic acid target regioncorresponding to the HPV33 E6 and/or E7 gene(s). In certain embodiments,(a) the first HPV33 amplification oligomer comprises a firsttarget-hybridizing sequence that is from about 15 to about 27 contiguousnucleotides in length and substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:66 andthat includes at least the sequence of SEQ ID NO:67, SEQ ID NO:68, orSEQ ID NO:69; and (b) the second HPV33 amplification oligomer comprisesa second target-hybridizing sequence that is from about 15 to about 27contiguous nucleotides in length and substantially corresponding to, oridentical to, a sequence that is contained in the sequence of SEQ IDNO:70 and that includes at least the sequence of SEQ ID NO:71, SEQ IDNO:72, or SEQ ID NO:73. In some embodiments of the oligomer combination,(a) the first HPV33 amplification oligomer comprises a firsttarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; and/or(b) the second HPV33 amplification oligomer comprises a secondtarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ IDNO:16. In typical variations, the oligomer combination is for detectingan HPV33 target nucleic acid in a sample suspected of containing HPV33and at least one other HPV genotype (e.g., at least one of HPV types 16,18, 31, 45, 52, and 58). In one variation, the combination of oligomersis a combination that includes at least two oligomers that aresubstantially identical to oligomers in Table 4.

In some preferred variations, the oligomer combination further includesfirst and second amplification oligomers for specifically amplifying anHPV type 31 (HPV31) nucleic acid target region corresponding to theHPV31 E6 and/or E7 gene(s). In certain embodiments, (a) the first HPV31amplification oligomer comprises a first target-hybridizing sequencethat is from about 15 to about 27 contiguous nucleotides in length andsubstantially corresponding to, or identical to, a sequence that iscontained in the sequence of SEQ ID NO:74 and that includes at least thesequence of SEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77; and (b) thesecond HPV31 amplification oligomer comprises a secondtarget-hybridizing sequence that is from about 15 to about 30 contiguousnucleotides in length and substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:78 andthat includes at least the sequence of SEQ ID NO:79, SEQ ID NO:80, orSEQ ID NO:81. In some embodiments of the oligomer combination, (a) afirst HPV31 amplification oligomer comprising a first target-hybridizingsequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; and/or (b) asecond HPV31 amplification oligomer comprising a secondtarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence selected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ IDNO:41.

In certain embodiments, an amplification oligomer as described herein isa promoter primer or promoter provider further comprising a promotersequence located 5′ to the target-hybridizing sequence and which isnon-complementary to the HPV33 or HPV31 target nucleic acid. Forexample, in some embodiments of an oligomer combination as describedherein for amplification of a HPV33 or HPV31 target region, the secondamplification oligomer is a promoter primer or promoter provider furthercomprising a 5′ promoter sequence. In particular embodiments, thepromoter sequence is a T7 RNA polymerase promoter sequence such as, forexample, a T7 promoter sequence having the sequence shown in SEQ IDNO:82. In specific variations, the second HPV33 amplification oligomeris a promoter primer or promoter provider having the sequence shown inSEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; and/or the second HPV31amplification oligomer is a promoter primer or promoter provider havingthe sequence shown in SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, or SEQ ID NO:49.

In some embodiments, an oligomer combination as described herein furtherincludes a terminating oligonucleotide (also referred to herein as a“blocker” oligonucleotide) comprising comprises a base sequencesubstantially complementary (e.g., fully complementary) to a sequencecontained within the target nucleic acid in the vicinity of the 5′-endof the target region. A terminating oligomer is typically used incombination with, e.g., a promoter provider amplification oligomer, suchas, for example, in certain embodiments described herein relating totranscription-mediated amplification (TMA).

In some embodiments, an oligomer combination as described herein furthercomprises at least one capture probe oligomer for capture of an HPV33and/or HPV31 target nucleic acid. Such capture probes may be specificfor either HPV33 or HPV31 target nucleic acid. In some embodiments, thecapture probe oligomer comprises a target-hybridizing sequencesubstantially corresponding to a sequence contained in the complement ofSEQ ID NO:83 (representative HPV33 E6/E7 region) or SEQ ID NO:84(representative HPV31 E6/E7 region), wherein the target-hybridizingsequence is covalently attached to a sequence or moiety that binds to animmobilized probe. In specific variations, the target-hybridizingsequence of an HPV33-specific capture probe oligomer comprises orconsists of a sequence substantially corresponding to, or identical to,SEQ ID NO:50; and/or the target hybridizing sequence of anHPV31-specific capture probe oligomer comprises or consists of asequence substantially corresponding to, or identical to, SEQ ID NO:52.Particularly suitable HPV33 or HPV31 capture probe oligomers for use inaccordance with the present invention comprise or consist of a sequenceas shown in SEQ ID NO:51 or SEQ ID NO:53, respectively.

In certain variations, an oligomer combination as described hereinfurther comprises at least one detection probe oligomer configured tospecifically hybridize to an HPV33 or HPV31 target sequence that isamplifiable using the first and second HPV33 or HPV31 amplificationoligomers (e.g., an HPV target sequence that is flanked by thetarget-hybridizing sequences of the first and second amplificationoligomers). Suitable HPV33 detection probes include oligomers comprisinga target-hybridizing sequence that is from about 14 to about 35nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:83 from about nucleotideposition 128 to about nucleotide position 164. For example, in somevariations, an HPV33 detection probe comprises a target-hybridizingsequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57,and SEQ ID NO:58. Suitable HPV31 detection probes include oligomerscomprising a target-hybridizing sequence that is from about 14 to about40 nucleotides in length and configured to specifically hybridize to atarget sequence contained within SEQ ID NO:84 from about nucleotideposition 675 to about nucleotide position 735. For example, in somevariations, an HPV31 detection probe comprises a target-hybridizingsequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65.

Typically, a detection probe oligomer in accordance with the presentinvention further includes a label. Particularly suitable labels includecompounds that emit a detectable light signal, e.g., fluorophores orluminescent (e.g., chemiluminescent) compounds that can be detected in ahomogeneous mixture. More than one label, and more than one type oflabel, may be present on a particular probe, or detection may rely onusing a mixture of probes in which each probe is labeled with a compoundthat produces a detectable signal (see, e.g., U.S. Pat. Nos. 6,180,340and 6,350,579, each incorporated by reference herein). Labels may beattached to a probe by various means including covalent linkages,chelation, and ionic interactions, but preferably the label iscovalently attached. For example, in some embodiments, a detection probehas an attached chemiluminescent label such as, e.g., an acridiniumester (AE) compound (see, e.g., U.S. Pat. Nos. 5,185,439; 5,639,604;5,585,481; and 5,656,744; each incorporated by reference herein), whichin typical variations is attached to the probe by a non-nucleotidelinker (see, e.g., U.S. Pat. Nos. 5,585,481; 5,656,744; and 5,639,604,particularly at column 10, line 6 to column 11, line 3, and Example 8;each incorporated by reference herein). In other embodiments, adetection probe comprises both a fluorescent label and a quencher, acombination that is particularly useful in fluorescence resonance energytransfer (FRET) assays. Specific variations of such detection probesinclude, e.g., a TaqMan detection probe (Roche Molecular Diagnostics)and a “molecular beacon” (see, e.g., Tyagi et al., Nature Biotechnol.16:49-53, 1998; U.S. Pat. Nos. 5,118,801 and 5,312,728; eachincorporated by reference herein).

A detection probe oligomer in accordance with the present invention mayfurther include a non-target-hybridizing sequence. Specific embodimentsof such detection probes include, for example, probes that formconformations held by intramolecular hybridization, such asconformations generally referred to as hairpins. Particularly suitablehairpin probes include a “molecular torch” (see, e.g., U.S. Pat. Nos.6,849,412; 6,835,542; 6,534,274; and 6,361,945, each incorporated byreference herein) and a “molecular beacon” (see, e.g., Tyagi et al.,supra; U.S. Pat. Nos. 5,118,801 and 5,312,728, supra). Methods for usingsuch hairpin probes are well known in the art.

In yet other embodiments, a detection probe is a linear oligomer thatdoes not substantially form conformations held by intramolecular bonds.In specific variations, a linear detection probe oligomer includes achemiluminescent compound as the label, preferably an acridinium ester(AE) compound.

Also provided by the present invention are detection probe oligomers andcapture probe oligomers as described herein.

In another aspect, the present invention provides methods for detectingan HPV33 target nucleic acid and/or HPV31 target nucleic acid in asample using an oligomer combination as described herein. In certainembodiments, a method for detecting an HPV33 target nucleic acid in asample suspected of containing HPV33 generally includes the followingsteps: (b) contacting the sample with an oligomer combination forspecifically amplifying an HPV33 nucleic acid target region, where theoligomer combination includes first and second HPV33 amplificationoligomers as described above; (c) performing an in vitro nucleic acidamplification reaction, where any HPV33 target nucleic acid present insaid sample is used as a template for generating an HPV33 amplificationproduct; and (d) detecting the presence or absence of the HPV33amplification product, thereby indicating the presence or absence ofHPV33 in the sample. In some embodiments, the sample is furthersuspected of containing at least one other HPV type (e.g., at least oneof HPV types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68;typically at least one of HPV types 31, 52, and 58), where the methodspecifically detects the presence or absence of HPV33 irrespective ofthe presence of the other HPV type(s) (e.g., the method is capable ofdistinguishing HPV33 from the other HPV types, such as at least one ofHPV types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68; typicallyat least one of HPV types 31, 52, and 58.)

In some variations, a method detecting an HPV31 target nucleic acid in asample suspected of containing HPV31 generally includes the followingsteps; (b′) contacting the sample with oligomer combination forspecifically amplifying an HPV31 nucleic acid target region, where theoligomer combination includes first and second HPV31 amplificationoligomers as described above; (c) performing an in vitro nucleic acidamplification reaction, where any HPV31 target nucleic acid present insaid sample is used as a template for generating an HPV31 amplificationproduct; and (d′) detecting the presence or absence of the HPV31amplification product, thereby indicating the presence or absence ofHPV31 in the sample. In some embodiments, the sample is furthersuspected of containing at least one other HPV type (e.g., at least oneof HPV types 16, 18, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68;typically at least one of HPV types 33, 52, and 58), where the methodspecifically detects the presence or absence of HPV31 irrespective ofthe presence of the other HPV type(s) (e.g., the method is capable ofdistinguishing HPV31 from the other HPV types, such as at least one ofHPV types 16, 18, 31, 35, 39, 45, 51, 52, 56, 58, 59, and 68; typicallyat least one of HPV types 33, 52, and 58.)

In some preferred embodiments, a detection method as above is fordetecting both HPV33 and HPV31 in a sample suspected of containing atleast one of HPV33 and HPV31. In some such embodiments, the methodincludes (I) steps (b)-(d) as above for detection of HPV33 and (II)steps (b′)-(d′) above for detecting HPV31. Any one or more of the stepsfor detecting HPV33 and HPV31 may be performed separately or together.In some variations, any one or more of steps (b)-(d) for HPV33 detectionare performed together with the one or more corresponding steps(b′)-(d′) for HPV31 detection in the same reaction mixture (e.g.,amplification step (c) may be performed with during amplification step(c) in the same reaction mixture; and/or detection step (d) may beperformed with detection step (d′) in the same reaction mixture).Accordingly, in some embodiments, the method for detecting HPV33 andHPV31 is performed as a multiplex assay for simultaneous detection ofboth HPV33 and HP31 within the same reaction mixture.

A detection method in accordance with the present invention typicallyfurther includes the step of (a) providing the sample suspected ofcontaining HPV33 and/or HPV31. In certain embodiments, “providing” asample to be used in steps (b)-(d) and/or steps (b′)-(d′) includes, forexample, receiving the sample at a testing facility or other locationwhere one or more steps of the method are performed, and/or retrievingthe sample from a location (e.g., from storage or other depository)within a facility where one or more steps of the method are performed.

In certain embodiments, the method further includes purifying the HPV33and/or HPV31 target nucleic acid from other components in the samplebefore the contacting step. Such purification may include may includemethods of separating and/or concentrating organisms contained in asample from other sample components. In particular embodiments,purifying the target nucleic acid includes capturing the target nucleicacid to specifically or non-specifically separate the target nucleicacid from other sample components. Non-specific target capture methodsmay involve selective precipitation of nucleic acids from asubstantially aqueous mixture, adherence of nucleic acids to a supportthat is washed to remove other sample components, or other means ofphysically separating nucleic acids from a mixture that contains HPVnucleic acid and other sample components.

In some embodiments, an HPV33 or HPV31 nucleic acid is selectivelyseparated from other sample components by specifically hybridizing theHPV target nucleic acid to a capture probe oligomer. The capture probeoligomer comprises a target-hybridizing sequence configured tospecifically hybridize to an HPV target sequence so as to form atarget-sequence:capture-probe complex that is separated from samplecomponents. Suitable HPV33 and HPV31 target-hybridizing sequences areshown in SEQ ID NO:50 and SEQ ID NO:52, respectively. In a preferredvariation, the specific target capture binds the HPVtarget:capture-probe complex to an immobilized probe to form atarget:capture-probeimmobilized-probe complex that is separated from thesample and, optionally, washed to remove non-target sample components(see, e.g., U.S. Pat. Nos. 6,110,678; 6,280,952; and 6,534,273; eachincorporated by reference herein). In such variations, the capture probeoligomer further comprises a sequence or moiety that binds the captureprobe, with its bound target sequence, to an immobilized probe attachedto a solid support, thereby permitting the hybridized target nucleicacid to be separated from other sample components. In embodimentscomprising amplification and detection of both HPV33 and HPV31,selective separation of HPV33 and HPV31 nucleic acids may be performedsimultaneously from a single sample.

In more specific embodiments, the capture probe oligomer includes a tailportion (e.g., a 3′ tail) that is not complementary to the HPV targetsequence but that specifically hybridizes to a sequence on theimmobilized probe, thereby serving as the moiety allowing the targetnucleic acid to be separated from other sample components, such aspreviously described in, e.g., U.S. Pat. No. 6,110,678, incorporatedherein by reference. Any sequence may be used in a tail region, which isgenerally about 5 to 50 nt long, and preferred embodiments include asubstantially homopolymeric tail of about 10 to 40 nt (e.g., A₁₀ toA₄₀), more preferably about 14 to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ toT₃A₃₀), that bind to a complementary immobilized sequence (e.g., poly-T)attached to a solid support, e.g., a matrix or particle. For example, inspecific embodiments of a capture probe comprising a 3′ tail, the HPV33or HPV31 capture probe oligomer has a sequence as shown in SEQ ID NO:51or SEQ ID NO:53, respectively.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probe oligomers that hybridize specificallyto the HPV target sequence under hybridizing conditions, usually at atemperature higher than the T_(m) of thetail-sequenceimmobilized-probe-sequence duplex. For embodimentscomprising a capture probe tail, the HPV-target:capture-probe complex iscaptured by adjusting the hybridization conditions so that the captureprobe tail hybridizes to the immobilized probe, and the entire complexon the solid support is then separated from other sample components. Thesupport with the attachedimmobilized-probe:capture-probe:HPV-target-sequence may be washed one ormore times to further remove other sample components. Preferredembodiments use a particulate solid support, such as paramagnetic beads,so that particles with the attachedHPV-target:capture-probe:immobilized-probe complex may be suspended in awashing solution and retrieved from the washing solution, preferably byusing magnetic attraction. To limit the number of handling steps, theHPV target nucleic acid may be amplified by simply mixing the HPV targetsequence in the complex on the support with amplification oligomers andproceeding with amplification steps.

Amplifying an HPV33 or HPV31 target sequence utilizes an in vitroamplification reaction using at least two amplification oligomers thatflank a target region to be amplified. In particular embodiments, theHPV33 target region to be amplified substantially corresponds to SEQ IDNO:83 from about nucleotide position 128 to about nucleotide position164. In particular embodiments, the HPV31 target region to be amplifiedsubstantially corresponds to SEQ ID NO:84 from about nucleotide position675 to about nucleotide position 735. Particularly suitableamplification oligomer combinations for amplification of these targetregions are described herein. Suitable amplification methods include,for example, replicase-mediated amplification, polymerase chain reaction(PCR), ligase chain reaction (LCR), strand-displacement amplification(SDA), and transcription-mediated or transcription-associatedamplification (TMA). Such amplification methods are well-known in theart and are readily used in accordance with the methods of the presentinvention.

For example, some amplification methods that use TMA amplificationinclude the following steps. Briefly, the target nucleic acid thatcontains the sequence to be amplified is provided as single strandednucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art willappreciate that conventional melting of double stranded nucleic acid(e.g., dsDNA) may be used to provide single-stranded target nucleicacids. A promoter primer binds specifically to the target nucleic acidat its target sequence and a reverse transcriptase (RT) extends the 3′end of the promoter primer using the target strand as a template tocreate a cDNA copy of the target sequence strand, resulting in anRNA:DNA duplex. An RNase digests the RNA strand of the RNA:DNA duplexand a second primer binds specifically to its target sequence, which islocated on the cDNA strand downstream from the promoter primer end. RTsynthesizes a new DNA strand by extending the 3′ end of the secondprimer using the first cDNA template to create a dsDNA that contains afunctional promoter sequence. An RNA polymerase specific for thepromoter sequence then initiates transcription to produce RNAtranscripts that are about 100 to 1000 amplified copies (“amplicons”) ofthe initial target strand in the reaction. Amplification continues whenthe second primer binds specifically to its target sequence in each ofthe amplicons and RT creates a DNA copy from the amplicon RNA templateto produce an RNA:DNA duplex. RNase in the reaction mixture digests theamplicon RNA from the RNA:DNA duplex and the promoter primer bindsspecifically to its complementary sequence in the newly synthesized DNA.RT extends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds totranscribe additional amplicons that are complementary to the targetstrand. The autocatalytic cycles of making more amplicon copies repeatduring the course of the reaction resulting in about a billion-foldamplification of the target nucleic acid present in the sample. Theamplified products may be detected in real-time during amplification, orat the end of the amplification reaction by using a probe that bindsspecifically to a target sequence contained in the amplified products.Detection of a signal resulting from the bound probes indicates thepresence of the target nucleic acid in the sample.

In some embodiments, the method utilizes a “reverse” TMA reaction. Insuch variations, the initial or “forward” amplification oligomer is apriming oligonucleotide that hybridizes to the target nucleic acid inthe vicinity of the 3′-end of the target region. A reverse transcriptase(RT) synthesizes a cDNA strand by extending the 3′-end of the primerusing the target nucleic acid as a template. The second or “reverse”amplification oligomer is a promoter primer or promoter provider havinga target-hybridizing sequence configured to hybridize to atarget-sequence contained within the synthesized cDNA strand. Where thesecond amplification oligomer is a promoter primer, RT extends the 3′end of the promoter primer using the cDNA strand as a template to createa second, cDNA copy of the target sequence strand, thereby creating adsDNA that contains a functional promoter sequence. Amplification thencontinues essentially as described above for initiation of transcriptionfrom the promoter sequence utilizing an RNA polymerase. Alternatively,where the second amplification oligomer is a promoter provider, aterminating oligonucleotide, which hybridizes to a target sequence thatis in the vicinity to the 5′-end of the target region, is typicallyutilized to terminate extension of the priming oligomer at the 3′-end ofthe terminating oligonucleotide, thereby providing a defined 3′-end forthe initial cDNA strand synthesized by extension from the primingoligomer. The target-hybridizing sequence of the promoter provider thenhybridizes to the defined 3′-end of the initial cDNA strand, and the3′-end of the cDNA strand is extended to add sequence complementary tothe promoter sequence of the promoter provider, resulting in theformation of a double-stranded promoter sequence. The initial cDNAstrand is then used a template to transcribe multiple RNA transcriptscomplementary to the initial cDNA strand, not including the promoterportion, using an RNA polymerase that recognizes the double-strandedpromoter and initiates transcription therefrom. Each of these RNAtranscripts is then available to serve as a template for furtheramplification from the first priming amplification oligomer.

Detection of the amplified products may be accomplished by a variety ofmethods. The nucleic acids may be associated with a surface that resultsin a physical change, such as a detectable electrical change. Amplifiednucleic acids may be detected by concentrating them in or on a matrixand detecting the nucleic acids or dyes associated with them (e.g., anintercalating agent such as ethidium bromide or cyber green), ordetecting an increase in dye associated with nucleic acid in solutionphase. Other methods of detection may use nucleic acid detection probesthat are configured to specifically hybridize to a sequence in theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481; each incorporated by referenceherein). Directly or indirectly labeled probes that specificallyassociate with the amplified product provide a detectable signal thatindicates the presence of the target nucleic acid in the sample. Forexample, if the target nucleic acid is the E6/E7 region of the HPV33genome or an RNA corresponding to the E6/E7 region, the amplifiedproduct will contain a target sequence in or complementary to a sequencein E6/E7 region, and a probe will bind directly or indirectly to asequence contained in the amplified product to indicate the presence ofthe target nucleic acid in the tested sample.

Preferred embodiments of detection probes that hybridize to thecomplementary amplified sequences may be DNA or RNA oligomers, oroligomers that contain a combination of DNA and RNA nucleotides, oroligomers synthesized with a modified backbone, e.g., an oligomer thatincludes one or more 2′-methoxy substituted ribonucleotides. Probes usedfor detection of the amplified HPV sequences may be unlabeled anddetected indirectly (e.g., by binding of another binding partner to amoiety on the probe) or may be labeled with a variety of detectablelabels. Particular embodiments of detection probes suitable for use inaccordance with methods of the present invention are further describedherein. In some preferred embodiments of the method for detecting HPVsequences, such as in certain embodiments using transcription-mediatedamplification (TMA), the detection probe is a linear chemiluminescentlylabeled probe, more preferably, a linear acridinium ester (AE) labeledprobe.

Oligomers that are not intended to be extended by a nucleic acidpolymerase preferably include a blocker group that replaces the 3′ OH toprevent enzyme-mediated extension of the oligomer in an amplificationreaction. For example, blocked amplification oligomers and/or detectionprobes present during amplification preferably do not have a functional3′ OH and instead include one or more blocking groups located at or nearthe 3′ end. A blocking group near the 3′ end is preferably within fiveresidues of the 3′ end and is sufficiently large to limit binding of apolymerase to the oligomer, and other preferred embodiments contain ablocking group covalently attached to the 3′ terminus. Many differentchemical groups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

Examples of oligomers that are typically blocked at the 3′ end—and whichare particularly suitable in certain embodiments usingtranscription-mediated amplification—are promoter providers. Asdescribed previously, a promoter provider comprises firsttarget-hybridizing region and, situated 5′ to the first region, a secondregion comprising a promoter sequence for an RNA polymerase. Thepromoter provider oligonucleotide is modified to prevent the initiationof DNA synthesis from its 3′-terminus, such as by including a blockergroup as discussed above. In some embodiments, a promoter provider foruse in accordance with an HPV33 detection method comprises atarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:70 andthat includes at least the sequence of SEQ ID NO:71, SEQ ID NO:72, orSEQ ID NO:73 (for example, a target-hybridizing sequence having asequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; e.g., an HPV33promoter provider may have a sequence as shown in SEQ ID NO:17, SEQ IDNO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ IDNO:23, or SEQ ID NO:24). In some embodiments, a promoter provider foruse in accordance with an HPV31 detection method comprises atarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:78 andthat includes at least the sequence of SEQ ID NO:79, SEQ ID NO:80, orSEQ ID NO:81 (for example, a target-hybridizing sequence having asequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; e.g., anHPV31 promoter provider may have a sequence as shown in SEQ ID NO:42,SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, or SEQ ID NO:49).

Another example of typically 3′-blocked oligomers are terminating(“blocker”) oligonucleotides, previously described above. A terminatingoligomer is typically used in combination with, e.g., a promoterprovider amplification oligomer, such as, for example, in certainembodiments described herein relating to transcription-mediatedamplification (TMA). A terminating oligomer hybridizes to a sequencecontained within the target nucleic acid in the vicinity of the 5′-endof the target region so as to “terminate” primer extension of a nascentnucleic acid that includes a priming oligonucleotide, thereby providinga defined 3′-end for the nascent nucleic acid strand.

Other embodiments using transcription-mediated amplification utilize apromoter primer, which comprises a first target-hybridizing region and,situated 5′ to the first region, a second region comprising a promotersequence for an RNA polymerase, but which is not modified to prevent theinitiation of DNA synthesis from its 3′-terminus. In certain variations,a promoter primer for use in accordance with an HPV33 detection methodcomprises a target-hybridizing sequence substantially corresponding to,or identical to, a sequence that is contained in the sequence of SEQ IDNO:70 and that includes at least the sequence of SEQ ID NO:71, SEQ IDNO:72, or SEQ ID NO:73 (for example, a target-hybridizing sequencehaving a sequence substantially corresponding to, or identical to, asequence selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; e.g.,an HPV33 promoter primer may have a sequence as shown in SEQ ID NO:17,SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,SEQ ID NO:23, or SEQ ID NO:24). In certain variations, a promoter primerfor use in accordance with an HPV31 detection method comprises atarget-hybridizing sequence substantially corresponding to, or identicalto, a sequence that is contained in the sequence of SEQ ID NO:78 andthat includes at least the sequence of SEQ ID NO:79, SEQ ID NO:80, orSEQ ID NO:81 (for example, a target-hybridizing sequence having asequence substantially corresponding to, or identical to, a sequenceselected from SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41; e.g., anHPV31 promoter primer may have a sequence as shown in SEQ ID NO:42, SEQID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, or SEQ ID NO:49).

Assays for detection of an HPV target nucleic acid may optionallyinclude a non-target internal control (IC) nucleic acid that isamplified and detected in the same assay reaction mixtures by usingamplification and detection oligomers specific for the IC sequence. ICnucleic acid sequences can be synthetic nucleic acid sequences that arespiked into a sample or the IC nucleic acid sequences may be a cellularcomponent. IC nucleic acid sequences that are cellular components can befrom exogenous cellular sources or endogenous cellular sources relativeto the specimen. An exogenous cellular source, for example, is a cellthat is added into the sample and that then flows through the sampleprocessing procedures along with the specimen. A more particular examplewould be the addition of a HeLa cell, Jurkat cell, SiHa cell or other tothe sample medium along with the specimen that is collected for testing(e.g., a vaginal swab specimen). The specimen and the exogenous cellsare then processed, amplified and detected, the specimen being amplifiedand detected using amplification and detection oligomers for identifyingthe target sequence of interest and the exogenous cells being amplifiedand detected using amplification and detection oligomers for identifyingan IC target sequence. An endogenous cellular source is a cellularsource that would naturally be obtained when gathering the specimen. Oneexample: epithelial cells will present when obtaining a specimen via avaginal swab. Similar then to the above exemplary exogenous cellsprocess described, the specimen and the endogenous cellular source areboth processed, amplified, and detected. The specimen being amplifiedand detected using amplification and detection oligomers for identifyingthe target sequence of interest and the endogenous cells being amplifiedand detected using amplification and detection oligomers for identifyingan IC target sequence; typically a housekeeping gene present in theendogenous cellular source, such as a beta-globulin gene. (See e.g.,Poljak et al., J. Clin. Virol, 25: S89-97, 2002; U.S. Pat. No.6,410,321; and US Patent Application Publication No. 2004-0023288; eachincorporated by reference herein). Use of a cellular source IC allowsfor a control from sample collection through detection. Syntheticnucleic acid sequences provide for control of amplification anddetection.

In certain embodiments, amplification and detection of a signal from theamplified IC sequence demonstrates that the assay reagents, conditions,and performance of assay steps were properly used in the assay if nosignal is obtained for the intended target HPV nucleic acid (e.g.,samples that test negative for the HPV33 and/or HPV31 target nucleicacid). An IC may also be used as an internal calibrator for the assaywhen a quantitative result is desired, i.e., the signal obtained fromthe IC amplification and detection is used to set a parameter used in analgorithm for quantitating the amount of HPV nucleic acid in a samplebased on the signal obtained for an amplified HPV target sequence. ICsare also useful for monitoring the integrity of one or more steps in anassay. A preferred embodiment of a synthetic IC nucleic acid sequence isa randomized sequence that has been derived from a naturally occurringsource (e.g., an HIV sequence that has been rearranged in a randommanner). Another preferred IC nucleic acid sequence may be an RNAtranscript isolated from a naturally occurring source or synthesized invitro, such as by making transcripts from a cloned randomized sequencesuch that the number of copies of IC included in an assay may beaccurately determined. The amplification oligomers and probe for the ICtarget sequence are configured and synthesized by using any well-knownmethod provided that the amplification oligomers and probe function foramplification of the IC target sequence and detection of the amplifiedIC sequence using substantially the same assay conditions used toamplify and detect the HPV target sequence. In preferred embodimentsthat include a target capture-based purification step, it is preferredthat a target capture probe specific for the IC target be included inthe assay in the target capture step so that the IC is treated in theassay in a manner analogous to that for the intended HPV analyte in allof the assay steps.

In some preferred embodiments, the detection of HPV33 and HPV31 isperformed as a multiplex assay. Typically, in such embodiments, theamplification of an HPV33 target nucleic acid is performedsimultaneously with the amplification of an HPV31 target nucleic acid inthe same amplification reaction mixture, and the detection of the HPV33amplification product is performed simultaneously with the detection ofthe HPV31 amplification product in the same detection reaction mixture.The detection of HPV33 and HPV31 typically includes contacting theamplification reaction with differentially labeled HPV33 and HPV31detection probe oligomers configured to specifically hybridize to theHPV33 and HPV31 amplification products under conditions whereby thepresence or absence of the HPV33 and HPV31 amplification products isdetermined. In some such variations, each of the HPV33 and HPV31detection probe oligomers comprises either a chemiluminescent label or afluorescent label. For example, each of the HPV33 and HPV31 detectionprobe oligomers may comprise a different chemiluminescent label, suchas, e.g., chemiluminescent labels characterized by different lightemission kinetics sufficient to distinguish between HPV33-specific andHPV31-specific chemiluminescent signals. Suitable chemiluminescentlabels for the HPV33 and HPV31 detection probe oligomers includeacridinium ester (AE) labels with differential light kinetics. Inparticular variations, one detection probe oligomer (e.g., a probespecific for an HPV33 amplification product) comprises an 2′ methylacridinium ester label (also referred to as a “glower” probe), while asecond detection probe oligomer (e.g., a probe specific for an HPV31amplification product) comprises an ortho fluoro acridinium ester label(also referred to herein has a “flasher” probe), the flasher probeexhibiting more rapid light-off kinetics than the glower probe. An ETFalgorithm may be used to deconvolute light-off kinetics and calculateeach signal. If using an internal control (IC), the IC probe may also,e.g., be a flasher probe, which can be distinguished from the HPV testflasher signal by, for example, using a much lower probe input than forthe HPV flasher probe. (See e.g., U.S. Pub. No. 2012/0003646).

Also provided by the subject invention is a reaction mixture foramplification and/or detection of an HPV33 and/or HPV31 target nucleicacid. A reaction mixture in accordance with the present invention atleast comprises one or more of the following: an oligomer combination asdescribed herein for amplification of an HPV33 and/or HPV31 targetnucleic acid; a capture probe oligomer as described herein for purifyingthe HPV33 and/or HPV31 target nucleic acid; and a detection probeoligomer as described herein for determining the presence or absence ofan HPV33 and/or HPV31 amplification product. The reaction mixture mayfurther include a number of optional components such as, for example,arrays of capture probe nucleic acids. For an amplification reactionmixture, the reaction mixture will typically include other reagentssuitable for performing in vitro amplification such as, e.g., buffers,salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP,dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes (e.g., reversetranscriptase, and/or RNA polymerase), and will typically include testsample components, in which an HPV33 and/or HPV31 target nucleic acidmay or may not be present. In addition, for a reaction mixture thatincludes a detection probe together with an amplification oligomercombination, selection of amplification oligomers and detection probeoligomers for a reaction mixture are linked by a common target region(i.e., the reaction mixture will include a probe that binds to asequence amplifiable by an amplification oligomer combination of thereaction mixture).

Also provided by the subject invention are kits for practicing themethods as described herein. A kit in accordance with the presentinvention at least comprises one or more of the following: anamplification oligomer combination as described herein for amplificationof an HPV33 and/or HPV31 target nucleic acid; a capture probe oligomeras described herein for purifying the HPV33 and/or HPV31 target nucleicacid; and a detection probe oligomer as described herein for determiningthe presence or absence of an HPV33 and/or HPV31 amplification product.The kits may further include a number of optional components such as,for example, arrays of capture probe nucleic acids. Other reagents thatmay be present in the kits include reagents suitable for performing invitro amplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase). Oligomers as described herein may be packaged in a varietyof different embodiments, and those skilled in the art will appreciatethat the invention embraces many different kit configurations. Forexample, a kit may include amplification oligomers for only one targetregion of an HPV genome, or it may include amplification oligomers formultiple HPV target regions. In addition, for a kit that includes adetection probe together with an amplification oligomer combination,selection of amplification oligomers and detection probe oligomers for akit are linked by a common target region (i.e., the kit will include aprobe that binds to a sequence amplifiable by an amplification oligomercombination of the kit). In certain embodiments, the kit furtherincludes a set of instructions for practicing methods in accordance withthe present invention, where the instructions may be associated with apackage insert and/or the packaging of the kit or the componentsthereof.

The invention is further illustrated by the following non-limitingexamples.

Example 1 HPV 31/33 Genotyping Assay

This example describes an exemplary HPV 31/33 genotyping assay inaccordance with the present invention. The assay of this example is alsoreferred to herein as the “APTIMA HPV 31/33 genotyping assay” or “APTIMA31/33 GT assay.”

Table 1 below lists all the oligomers used in this assay at theirrespective concentrations.

TABLE 1 Oligomers used in HPV 31/33 genotyping assay Oligo descriptionOligo SEQ ID NO Concentration HPV31 target capture oligo 53 0.9 pmol/rxnHPV33 target capture oligo 51 0.7 pmol/rxn HPV31 T7 amp oligo 42 6.6pmol/rxn HPV33 T7 amp oligo 20 5 pmol/rxn HPV31 non-T7 amp oligo 29 6.7pmol/rxn HPV33 non-T7 amp oligo 5 6.6 pmol/rxn HPV33 detection probe 541.3 × 10⁶ RLU/ml HPV31 detection probe 59 8.5 × 10⁶ RLU/ml

This assay utilized methods and reagents from the APTIMA® HPV assay (seee.g., APTIMA HPV Assay, Cat. No. 303012, Gen-Probe Incorporated, SanDiego, Calif.). Generally, the testing was performed using a DTS 402instrument system and various APTIMA reagents according to the APTIMAassay package insert. A target capture reaction mixture was prepared tocontain target capture oligos SEQ ID NO53 and SEQ ID NO:51, which areconfigured to specifically hybridize HPV31 and HPV33 target nucleicacids, respectively. An amplification reaction mixture was prepared tocontain SEQ ID NOS: 5, 20, 29 and 42 as amplification oligomers. Thedetection reaction mixture was prepared to contain SEQ ID NOS: 54 and 59as AE labeled detection probe oligomers configured to specificallyhybridize amplification product generated from HPV31 and HPV 33,respectively.

Capture, amplification and detection reactions were performed asmultiplex reactions. HPV positive samples included HPV negative ThinPrepliquid pap samples spiked with various concentrations of HPV31 in vitrotranscripts (IVT), with HPV33 IVT, or with HPV31 and HPV33 IVT; and HPVpositive samples. Negative control sample were HPV negative ThinPrepliquid pap samples without any HPV IVT spiked therein. HPV positive orHPV negative was determined using the APTIMA® HPV assay (cat no. 303012,Gen-Probe Incorproated). Samples were diluted with sample transportmedia (STM) at a ratio of 1:2.9, and then a target capture reaction wasperformed. Reaction mixtures were incubated for 35 minutes at 62° C.,followed by 30 minutes at room temperature. The Gen-Probe SB100 systemwas used for all incubation steps. A capture and wash step was performedusing magnetic beads and the Gen-Probe Target Capture System (cat no.104555, Gen-Probe Incorporated). Following target capture, theamplification reaction mixture and oil was added to each reaction tubeand incubated for 10 minutes at 62° C. for primer anneal. Following the10 minute incubation, enzyme reagent was added to each reaction mixtureand the reactions were incubated for 60 minutes at 42° C. to allow fornucleic acid amplification of target nucleic acids. Followingamplification a detection reaction mixture was added to eachamplification reaction and incubated for 20 minutes at 62° C. to allowfor probe to hybridize to amplification product in the reactions. Aselection reagent was added to the reaction tube and incubated for 10minutes at 62° C. followed by cooling at room temperature. Detection oftarget was performed using the Gen-Probe Luminometer with Auto Detect 1and Auto Detect 2 reagents. Detection of amplification products wasmeasured in Relative Light Units (RLU). In general, for determiningpresence of target, an RLU cutoff value of 200,000 RLU was used forHPV31 and 800,000 RLU was used for HPV33

Analytical Sensitivity and Specificity

Very good analytical sensitivity was observed for both HPV types 31 and33 for IVT spiked into negative clinical samples. 100% positivity wasobtained for both HPV types at concentrations as low as 10copies/reaction.

Analytical sensitivity at also analyzed for both targets in the presenceof the other target. HPV31 IVT and HPV33 IVT were detected at 30 copiesper reaction in the presence of 1,000,000 copies per reaction of each ofHPV types 6, 11, 16, 18, 35, 39, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59,61, 66, 68, 71, and 81, and the results showed that both HPV31 and HPV33targets were efficiently amplified in the presence of these othertargets without cross-reactivity.

Cross-reactivity was further analyzed. Negative clinical pools werespiked with 1,000,000 copies per reaction of either HPV 52, HPV58 orboth HPV52 and HPV58. HPV 31 and HPV33 amplification and detectionreactions were performed as generally described above. No signal wasobserved with each of these samples. Similar experiments were set up byspiking a negative clinical pool with 1,000,000 copies per reaction ofseveral other HPV transcripts at 1,000,000 copies/reaction each. HPV 31and HPV33 amplification and detection reactions were performed asgenerally described above. No signal was observed with each of thesesamples. These data show that there is no cross-reactivity with theseother HPV types and that the assay is very specific to HPV 31 and 33.

Clinical Sensitivity and Specificity

Clinical specimens (n=137) were tested in the HPV31/33 genotyping assayto assess clinical sensitivity and specificity. HPV DNA genotyping wasperformed with the LINEAR ARRAY HPV Genotyping Test (LA, Roche MolecularDiagnostics). This test was used as the standard to determine if asample contained HPV31 and/or HPV33. Only a limited number of sampleswere available that tested HPV31 DNA positive (n=13) or HPV33 DNApositive (n=13).

Overall, with this limited data set, the clinical sensitivity fordetection of HVP31 was 84.6%. Two specimens were negative in the HPV31/33 genotyping assay that tested positive for HPV31 DNA in the LAassay. Both specimens contained HPV31 DNA along with additional HPV lowrisk types. Both specimens tested negative using the APTIMA HPVScreening assay, making it very unlikely that the specimens containedany HPV31 mRNA.

The clinical sensitivity for detection of HPV33 was 92.3%. One specimenwas negative in the HPV 31/33 genotyping assay that tested positive forHPV33 DNA in the LA assay. This specimen contained several other highand low risk HPV DNA types. The specimen tested negative using theAPTIMA HPV Screening assay and the HC2 test (Qiagen, Gaithersburg, Md.),indicating that it was unlikely this sample contained any HPV33 mRNA.

Clinical specificity for HPV31 was 98.4%. Two (2) specimens that wereHPV31 negative according to the assay LA tested positive using theHPV31/33 genotyping assay. Both specimens contained additional HPV highrisk types and tested positive using the APTIMA HPV Screening assay andthe HC2 test (Qiagen). Specificity for HPV33 was 100%.

A summary of the clinical specimen testing results is shown in Tables 2and 3 below.

TABLE 2 Results of clinical specimen testing for HPV31 HPV31 accordingto LA assay Positive Negative Total HPV31 Positive 11 2 13 according toNegative 2 122 124 APTIMA 31/33 Total 13 124 137 GT assay

TABLE 3 Results of clinical specimen testing for HPV33 HPV33 accordingto LA assay Positive Negative Total HPV33 Positive 12 0 12 according toNegative 1 124 125 APTIMA 31/33 Total 13 124 137 GT assay

SEQUENCES

TABLE 4 Exemplary Oligomer Sequences, Reference Sequences, and RegionsSEQ ID NO:  Sequence (5′→3′) Description 1 TCAAGACACTGAGGAAAAACCHPV33 Non-T7 amp oligo 2 GTTTCAAGACACTGAGGA HPV33 Non-T7 amp oligo 3TATGTTTCAAGACACTGAGGA HPV33 Non-T7 amp oligo 4 ACGACTATGTTTCAAGACACTGAGHPV33 Non-T7 amp oligo 5 GACTATGTTTCAAGACACTGAG HPV33 Non-T7 amp oligo 6CTGCACGACTATGTTTCAAG HPV33 Non-T7 amp oligo 7 GTACTGCACGACTATGTTTCAAGAHPV33 Non-T7 amp oligo 8 AGTAAGGTACTGCACGACT HPV33 Non-T7 amp oligo 9TAGTTGTCTCCAATGCTTG Target hybridizing sequence (THS) of SEQ ID NO: 1710 ATGTTGTGTATAGTTGTCTCC Target hybridizing sequence (THS) of SEQID NO: 18 11 TAGTTCAATGTTGTGTATAGTTGT Target hybridizingsequence (THS) of SEQ ID NO: 19 12 CTGTAGTTCAATGTTGTGTATAGTTGTarget hybridizing sequence (THS) of SEQ ID NO: 20 13GCACTGTAGTTCAATGTTGTG Target hybridizing sequence (THS) of SEQ ID NO: 2114 CACGCACTGTAGTTCAATG Target hybridizing sequence (THS) of SEQID NO: 22 15 CATTCCACGCACTGTAGTTCA Target hybridizingsequence (THS) of SEQ ID NO: 23 16 TGCATTCCACGCACTGTAGTTCATarget hybridizing sequence (THS) of SEQ ID NO: 24 17AATTTAATACGACTCACTATAGGGAGATAGTTGTCTCCAA HPV33 T7 amp oligo TGCTTG 18AATTTAATACGACTCACTATAGGGAGAATGTTGTGTATAG HPV33 T7 amp oligo TTGTCTCC 19AATTTAATACGACTCACTATAGGGAGATAGTTCAATGTTG HPV33 T7 amp oligo TGTATAGTTGT20 AATTTAATACGACTCACTATAGGGAGACTGTAGTTCAATG HPV33 T7 amp oligoTTGTGTATAGTTG 21 AATTTAATACGACTCACTATAGGGAGAGCACTGTAGTTCAHPV33 T7 amp oligo ATGTTGTG 22 AATTTAATACGACTCACTATAGGGAGACACGCACTGTAGTHPV33 T7 amp oligo TCAATG 23 AATTTAATACGACTCACTATAGGGAGACATTCCACGCACTHPV33 T7 amp oligo GTAGTTCA 24 AATTTAATACGACTCACTATAGGGAGATGCATTCCACGCAHPV33 T7 amp oligo CTGTAGTTCA 25 ATGAGCAATTACCCGACAGCHPV31 Non-T7 amp oligo 26 GAGCAATTACCCGACAGC HPV31 Non-T7 amp oligo 27TACCCGACAGCTCAGATGA HPV31 Non-T7 amp oligo 28 ACCCGACAGCTCAGATGAGGHPV31 Non-T7 amp oligo 29 GACAGCTCAGAGGAGGAGGATG HPV31 Non-T7 amp oligo30 GCTCAGATGAGGAGGATGTC HPV31 Non-T7 amp oligo 31 CAGATGAGGAGGATGTCATAHPV31 Non-T7 amp oligo 32 ATGAGGAGGATGTCATAGACA HPV31 Non-T7 amp oligo33 AGGAGGATGTCATAGACAGTC HPV31 Non-T7 amp oligo 34CACACAAACGAAGTGTAGACTTACACTGAC Target hybridizing sequence (THS) of SEQID NO: 42 35 GTGTAGACTTACACTGACAACA Target hybridizingsequence (THS) of SEQ ID NO: 43 36 CGAAGTGTAGACTTACACTGACATarget hybridizing sequence (THS) of SEQ ID NO: 44 37CAAACGAAGTGTAGACTTACAC Target hybridizing sequence (THS) of SEQID NO: 45 38 CACACAAACGAAGTGTAGACT Target hybridizingsequence (THS) of SEQ ID NO: 46 39 CTCTGTACACACAAACGAAGTTarget hybridizing sequence (THS) of SEQ ID NO: 47 40GTGTGCTCTGTACACACAAACG Target hybridizing sequence (THS) of SEQID NO: 48 41 GAATATCTACTTGTGTGCTCTG Target hybridizingsequence (THS) of SEQ ID NO: 49 42AATTTAATACGACTCACTATAGGGAGACACACAAACGAAG HPV31 T7 amp oligoTGTAGACTTACACTGAC 43 AATTTAATACGACTCACTATAGGGAGAGTGTAGACTTACAHPV31 T7 amp oligo CTGACAACA 44 AATTTAATACGACTCACTATAGGGAGACGAAGTGTAGACTHPV31 T7 amp oligo TACACTGACA 45AATTTAATACGACTCACTATAGGGAGACAAACGAAGTGTA HPV31 T7 amp oligo GACTTACAC 46AATTTAATACGACTCACTATAGGGAGACACACAAACGAAG HPV31 T7 amp oligo TGTAGACT 47AATTTAATACGACTCACTATAGGGAGACTCTGTACACACA HPV31 T7 amp oligo AACGAAGT 48AATTTAATACGACTCACTATAGGGAGAGTGTGCTCTGTAC HPV31 T7 amp oligo ACACAAACG 49AATTTAATACGACTCACTATAGGGAGAGAATATCTACTTG HPV31 T7 amp oligo TGTGTGCTC 50AAAUGUUUGCUUUAUAUAUGCACC Target hybridizing sequence (THS) of SEQID NO: 51 51 AAAUGUUUGCUUUAUAUAUGCACCTTTAAAAAAAAAAAAAHPV33 Target capture AAAAAAAAAAAAAAAAA oligo 52GCUCAUAACAGUGGAGGUCAGUUGCCUC Target hybridizing sequence (THS) of SEQID NO: 53 53 GCUCAUAACAGUGGAGGUCAGUUGCCUCtttaaaaaaaaaHPV31 Target capture aaaaaaaaaaaaaaaaaaaaa oligo 54 CCACGAACAUUGCAUGAUUUHPV33 Detection probe 55 CCACGAACAUUGCAUGAUUUGUG HPV33 Detection probe56 CGAACAUUGCAUGAUUUGUGC HPV33 Detection probe 57CCACGAACAUUGCAUGAUUUGUGCC HPV33 Detection probe 58CGAACAUUGCAUGAUUUGUGCC HPV33 Detection probe 59 CAGCUGGACAAGCAGAACCGGACHPV31 Detection probe 60 AGCUGGACAAGCAGAACCGGAC HPV31 Detection probe 61AGCUGGACAAGCAGAACCGGACA HPV31 Detection probe 62CAGCUGGACAAGCAGAACCGGACA HPV31 Detection probe 63 CUGGACAAGCAGAACCGGACHPV31 Detection probe 64 CUGGACAAGCAGAACCGGACACAUC HPV31 Detection probe65 CUGGACAAGCAGAACCGGACACAUCC HPV31 Detection probe 66AGTAAGGTACTGCACGACTATGTTTCAAGACACTGAGGAA Amp oligo hybridizing AAACCregion 67 GTTTCAAG Amp oligo core hybridizing sequence 68 ACACTGAGAmp oligo core hybridizing sequence 69 CTGCACGACT Amp oligo corehybridizing sequence 70 TGCATTCCACGCACTGTAGTTCAATGTTGTGTATAGTTGTAmp oligo hybridizing CTCCAATGCTTG region 71 CTGTAGTTC Amp oligo corehybridizing sequence 72 ATGTTGTG Amp oligo core hybridizing sequence 73AGTTGTCTCC Amp oligo core hybridizing sequence 74ATGAGCAATTACCCGACAGCTCAGAKGAGGAGGATGTCAT Amp oligo hybridizing AGACAGTCregion 75 AGGAGGATG Amp oligo core hybridizing sequence 76 CAGAKGAGGAmp oligo core hybridizing sequence 77 ACCCGACAGC Amp oligo corehybridizing sequence 78 GAATATCTACTTGTGTGCTCTGTACACACAAACGAAGTGTAmp oligo hybridizing AGACTTACACTGACAACA region 79 GTGTAGACTAmp oligo core hybridizing sequence 80 CACACAAACG Amp oligo corehybridizing sequence 81 TGTGCTCTG Amp oligo core hybridizing sequence 82AATTTAATACGACTCACTATAGGGAGA T7 promoter sequence 83GTAAACTATAATGCCAAGTTTTAAAAAAGTAGGGTGTAAC HPV33 E6/E7 referenceCGAAAGCGGTTCAACCGAAAACGGTGCATATATAAAGCAA sequenceACATTTTGCAGTAAGGTACTGCACGACTATGTTTCAAGACACTGAGGAAAAACCACGAACATTGCATGATTTGTGCCAAGCATTGGAGACAACTATACACAACATTGAACTACAGTGCGTGGAATGCAAAAAACCTTTGCAACGATCTGAGGTATATGATTTTGCATTTGCAGATTTAACAGTTGTATATAGAGAGGGAAATCCATTTGGAATATGTAAACTGTGTTTGCGGTTCTTATCTAAAATTAGTGAATATAGACATTATAATTATTCTGTATATGGAAATACATTAGAACAAACAGTTAAAAAACCTTTAAATGAAATATTAATTAGGTGTATTATATGTCAAAGACCTTTGTGTCCTCAAGAAAAAAAACGACATGTGGATTTAAACAAACGATTTCATAATATTTCGGGTCGTTGGGCAGGGCGCTGTGCGGCGTGTTGGAGGTCCCGACGTAGAGAAACTGCACTGTGACGTGTAAAAACGCCATGAGAGGACACAAGCCAACGTTAAAGGAATATGTTTTAGATTTATATCCTGAACCAACTGACCTATACTGCTATGAGCAATTAAGTGACAGCTCAGATGAGGATGAAGGCTTGGACCGGCCAGATGGACAAGCACAACCAGCCACAGCTGATTACTACATTGTAACCTGTTGTCACACTTGTAACACCACAGTTCGTTTATGTGTCAACAGTACAGCAAGTGACCTACGAACCATACAGCAACTACTTATGGGCACAGTGAATATTG TGTGCCCTACCTGTGCACAACAATAA 84TAATAATAATAATCTTAGTATAAAAAAGTAGGGAGTGACC HPV31 E6/E7 referenceGAAAGTGGTGAACCGAAAACGGTTGGTATATAAAGCACAT sequenceAGTATTTTGTGCAAACCTACAGACGCCATGTTCAAAAATCCTGCAGAAAGACCTCGGAAATTGCATGAACTAAGCTCGGCATTGGAAATACCCTACGATGAACTAAGATTGAATTGTGTCTACTGCAAAGGTCAGTTAACAGAAACAGAGGTATTAGATTTTGCATTTACAGATTTAACAATAGTATATAGGGACGACACACCACACGGAGTGTGTACAAAATGTTTAAGATTTTATTCAAAAGTAAGTGAATTTAGATGGTATAGATATAGTGTGTATGGAACAACATTAGAAAAATTGACAAACAAAGGTATATGTGATTTGTTAATTAGGTGTATAACGTGTCAAAGACCGTTGTGTCCAGAAGAAAAACAAAGACATTTGGATAAAAAGAAACGATTCCACAACATAGGAGGAAGGTGGACAGGACGTTGCATAGCATGTTGGAGAAGACCTCGTACTGAAACCCAAGTGTAAACATGCGTGGAGAAACACCTACGTTGCAAGACTATGTGTTAGATTTGCAACCTGAGGCAACTGACCTCCACTGTTATGAGCAATTACCCGACAGCTCAGATGAGGAGGATGTCATAGACAGTCCAGCTGGACAAGCAGAACCGGACACATCCAATTACAATATCGTTACCTTTTGTTGTCAGTGTAAGTCTACACTTCGTTTGTGTGTACAGAGCACACAAGTAGATATTCGCATATTGCAAGAGCTGTTAATGGGCTCATTTGGAATCGTGTGCCCCAACTG TTCTACTAGACTGTAA 85AGGAGGATGTCATAGACAAGTC HPV31 Non-T7 amp oligo 86AATTTAATACGACTCACTATAGGGAGAGAATATCTACTTG HPV31 T7 amp oligo TGTGCTC 87GAATATCTACTTGTGTGCTC Target hybridizing sequence (THS) of SEQ ID NO: 8688 gctcataacagtggaggtcagttgcctctttaaaaaaaaa HPV Target Captureaaaaaaaaaaaaaaaaaaaaa oligomer 89ctccaacacgctgcacagcgccctgtttaaaaaaaaaaaa HPV Target Captureaaaaaaaaaaaaaaaaaa oligomer 90 gtgcacagatcaggtagcttgtagggtcgtttaaaaaaaaHPV Target Capture aaaaaaaaaaaaaaaaaaaaaa oligomer 91GCTCATAACAGTGGAGGTCAGTTGCCTCTTTAAAAAAAAA HPV Target CaptureAAAAAAAAAAAAAAAAAAAAA oligomer 92 GCTCATAACAGTGGAGGTCAGTTGCCTCTarget hybridizing sequence (THS) of SEQ ID NO: 91 93GUGCACAGAUCAGGUAGCUUGUAGGGUCGTTTAAAAAAAA HPV Target CaptureAAAAAAAAAAAAAAAAAAAAAA oligomer 94 GUGCACAGAUCAGGUAGCUUGUAGGGUCGTarget hybridizing sequence (THS) of SEQ ID NO: 93 95ctccaacacgctgcacagcgccctg Target hybridizing sequence (THS) of SEQID NO: 89 96 gtgcacagatcaggtagcttgtagggtcg Target hybridizingsequence (THS) of SEQ ID NO: 90 97 gctcataacagtggaggtcagttgcctcTarget hybridizing sequence (THS) of SEQ ID NO: 88 98TTAARTGACAGCTCAGAKGAGGAKGATGAAATAGATGGTC Amp oligo hybridizingregion (HPV16) 99 CTCAGAKGAGGAKG Amp oligo core hybridizing region(HPV16) 100 GTGACAGCTCAGATGAGGATG HPV 16 Amp oligo 101GACAGCTCAGAKGAGGAGGATG HPV 16 Amp oligo 102 CTCAGAKGAGGAKGATGAAATAGATGGHPV 16 Amp oligo 103 RTGACAGCTCAGAKGAGGAKGATG HPV 16 Amp oligo 104GACAGCTCAGATGAGGAGGATG HPV 16 Amp oligo 105TGACAGCTCAGAKGAGGAKGATGAAATAG HPV 16 Amp oligo 106CAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTT Amp oligo hybridizingCGGTTGTGCG region (HPV16) 107 AGAGTCACACTTG Amp oligo corehybridizing region (HPV16) 108 AATTTAATACGACTCACTATAGGGAGAACTTGCAACAAAAHPV16 Amp Oligo GGTTAC 109 AATTTAATACGACTCACTATAGGGAGAACACTTGCAACAAHPV16 Amp Oligo AAGGTTACAATATTG 110AATTTAATACGACTCACTATAGGGAGAAGAGTCACACTTG HPV16 Amp Oligo CAACAAAAG 111AATTTAATACGACTCACTATAGGGAGAAGAGTCACACTTG HPV16 Amp Oligo CAACAAAAGG 112AATTTAATACGACTCACTATAGGGAGAGCACAACCGAAGC HPV16 Amp Oligo GTAGAGTC 113AATTTAATACGACTCACTATAGGGAGACGCACAACCGAAG HPV16 Amp OligoCGTAGAGTCACACTTGC 114 AATTTAATACGACTCACTATAGGGAGAAAGCGTAGAGTCAHPV16 Amp Oligo CACTTGC 115 AATTTAATACGACTCACTATAGGGAGAGCAACAAAAGGTTHPV16 Amp Oligo ACAATATTG 116 AATTTAATACGACTCACTATAGGGAGAGAAGCGTAGAGTCHPV16 Amp Oligo ACACTTG 117 CAACAAAAGGTTAC Amp oligo corehybridizing region (HPV16) 118 ACTTGCAACAAAAGGTTAC Target hybridizingsequence (THS) of SEQ ID NO: 104 119 ACACTTGCAACAAAAGGTTACAATATTGTarget hybridizing sequence (THS) of SEQ ID NO: 105 120AGAGTCACACTTGCAACAAAAG Target hybridizing sequence (THS) of SEQID NO: 106 121 AGAGTCACACTTGCAACAAAAGG Target hybridizingsequence (THS) of SEQ ID NO: 107 122 GCACAACCGAAGCGTAGAGTCTarget hybridizing sequence (THS) of SEQ ID NO: 108 123CGCACAACCGAAGCGTAGAGTCACACTTGC Target hybridizing sequence (THS) of SEQID NO: 109 124 AAGCGTAGAGTCACACTTGC Target hybridizingsequence (THS) of SEQ ID NO: 110 125 GCAACAAAAGGTTACAATATTGTarget hybridizing sequence (THS) of SEQ ID NO: 111 126GAAGCGTAGAGTCACACTTG Target hybridizing sequence (THS) of SEQ ID NO: 112127 CAGCKGGACAAGCAGAACCGGAC Detection probe (HPV16) 128 CGACGAGCCGAACCACHPV18 Amp Oligo 129 CGACGAGCCGAACCACA HPV18 Amp Oligo 130GACGAGCCGAACCACA HPV18 Amp Oligo 131 CGACGAGCCGAACCACAA HPV18 Amp Oligo132 GACGAGCCGAACCACAA HPV18 Amp Oligo 133 ACGAGCCGAACCACAAHPV18 Amp Oligo 134 AATTTAATACGACTCACTATAGGGAGAGTTCAGAAACAGCHPV18 Amp Oligo TGCTGG 135 AATTTAATACGACTCACTATAGGGAGAGTGTTCAGAAACAHPV18 Amp Oligo GCTGCTGG 136 AATTTAATACGACTCACTATAGGGAGAGGGTGTTCAGAAAHPV18 Amp Oligo CAGCTGCTGG 137 AATTTAATACGACTCACTATAGGGAGAGGGTGTTCAGAAAHPV18 Amp Oligo CAGCTG 138 AATTTAATACGACTCACTATAGGGAGAACACACAAAGGACHPV18 Amp Oligo AGGGT 139 AATTTAATACGACTCACTATAGGGAGAGACACACAAAGGAHPV18 Amp Oligo CAGGGT 140 AATTTAATACGACTCACTATAGGGAGAGCACACCACGGACHPV18 Amp Oligo ACACAAAGG 141 AATTTAATACGACTCACTATAGGGAGACACACCACGGACAHPV18 Amp Oligo CACAAAGGAC 142 AATTTAATACGACTCACTATAGGGAGACACACCACGGACAHPV18 Amp Oligo CACAAAG 143 CCAGCAGCTGTTTCTGAACACCCTGTCCTTTGTGTGTCCGHyb region 18 TGGTGTGC 144 GTTCAGAAACAGCTGCTGG Target hybridizingsequence (THS) of SEQ ID NO: 134 145 GTGTTCAGAAACAGCTGCTGGTarget hybridizing sequence (THS) of SEQ ID NO: 135 146GGGTGTTCAGAAACAGCTGCTGG Target hybridizing sequence (THS) of SEQID NO: 136 147 GGGTGTTCAGAAACAGCTG Target hybridizingsequence (THS) of SEQ ID NO: 137 148 ACACACAAAGGACAGGGTTarget hybridizing sequence (THS) of SEQ ID NO: 138 149GACACACAAAGGACAGGGT Target hybridizing sequence (THS) of SEQ ID NO: 139150 GCACACCACGGACACACAAAGG Target hybridizing sequence (THS) of SEQID NO: 140 151 CACACCACGGACACACAAAGGAC Target hybridizingsequence (THS) of SEQ ID NO: 141 152 CACACCACGGACACACAAAGTarget hybridizing sequence (THS) of SEQ ID NO: 142 153GCAGACGACCUUCGAGCAUUC Detection probe (HPV18) 154 CGUCUGCUGAGCUUUCUADetection probe (HPV18) 155 GUAGUAGAAAGCUCAGCAGACGACCDetection probe (HPV18) 156 GUAGAAACCUCGC Detection probe (HPV18) 157GUAGAGAGCUCGGCAGANGAC Detection probe (HPV18) 158 GUGUGACGGCAGAAUUGAGCDetection probe (HPV18) 159 UGUGUGUGUGUUGUAAGUGU Detection probe (HPV18)160 UCUUCUGCCGAGCUC Detection Probe 161 GUAGAGAGCUCGGCAGAGGACDetection Probe

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. An oligomer combination comprising at least oneHPV33 detection probe oligomer comprising (a) a target-hybridizingsequence that is from about 14 to about 35 nucleotides in length and isconfigured to specifically hybridize to a target sequence containedwithin SEQ ID NO:83 from about nucleotide position 128 to aboutnucleotide position 164, and (b) a chemiluminescent or fluorescentlabel.
 2. The oligomer combination of claim 1, wherein the HPV33detection probe target-hybridizing sequence is selected from the groupconsisting of SEQ ID NOs:54-58.
 3. An oligomer combination comprising atleast one HPV31 detection probe oligomer comprising (a) atarget-hybridizing sequence that is from about 14 to about 40nucleotides in length and is configured to specifically hybridize to atarget sequence contained within SEQ ID NO:84 from about nucleotideposition 675 to about nucleotide position 735, and (b) achemiluminescent or fluorescent label.
 4. The oligomer combination ofclaim 3, wherein the HPV31 detection probe target-hybridizing sequenceis selected from the group consisting of SEQ ID NOs:59-65.
 5. A methodfor detecting a human papillomavirus type 33 (HPV33) target nucleic acidin a sample suspected of containing HPV33 and at least one of HPV types31, 52, and 58, said method comprising: (a) providing a sample, whereinsaid sample is suspected of containing HPV33 and at least one of HPVtypes 31, 52, and 58; (b) contacting the sample with an oligomercombination for specifically amplifying an HPV33 nucleic acid targetregion, said oligomer combination comprising (i) a first HPV33amplification oligomer and (ii) a second HPV33 amplification oligomer,wherein said HPV33 nucleic acid target region comprises a sequencecontained within SEQ ID NO:83 from about nucleotide position 128 toabout nucleotide position 164; (c) performing an in vitro nucleic acidamplification reaction, wherein any HPV33 target nucleic acid present insaid sample is used as a template for generating an HPV33 amplificationproduct; and (d) detecting the presence or absence of the HPV33amplification product, wherein the detection step (d) comprisescontacting the amplification reaction of step (c) with an HPV33detection probe oligomer comprising a target-hybridizing sequence thatis from about 14 to about 35 nucleotides in length and is configured tospecifically hybridize to the target sequence contained within SEQ IDNO:83 from about nucleotide position 128 to about nucleotide position164, wherein said HPV detection probe oligomer is configured tospecifically hybridize to the HPV33 amplification product underconditions whereby the presence or absence of the HPV33 amplificationproduct is determined, thereby indicating the presence or absence ofHPV33 in said sample.
 6. The method of claim 5, wherein said method isfor further detecting an HPV type 31 (HPV31) target nucleic acid in saidsample, and wherein the method further comprises: (b′) contacting thesample with an oligomer combination for specifically amplifying an HPV31nucleic acid target region, said oligomer combination comprising (i) afirst HPV31 amplification oligomer and (ii) a second HPV31 amplificationoligomer; (c′) performing an in vitro nucleic acid amplificationreaction, wherein any HPV31 target nucleic acid present in said sampleis used as a template for generating an HPV31 amplification product; and(d′) detecting the presence or absence of the HPV31 amplificationproduct, wherein the detection step (d′) comprises contacting theamplification reaction of step (c′) with an HPV31 detection probeoligomer configured to specifically hybridize to the HPV31 amplificationproduct under conditions whereby the presence or absence of the HPV31amplification product is determined, thereby indicating the presence orabsence of HPV31 in said sample.
 7. The method of claim 5, wherein theHPV33 detection probe target-hybridizing sequence is selected from thegroup consisting of SEQ ID NOs:54-58.
 8. The method of claim 5, whereinthe HPV33 detection probe comprises a label selected from the groupconsisting of (a) a chemiluminescent label; (b) a fluorescent label; (c)a quencher; and (d) a combination of two or more of (a), (b), and (c).9. The method of claim 5, wherein the detection step (d) occurs duringthe amplification step (c).
 10. The method of claim 9, wherein the HPV33detection probe is a TaqMan detection probe, a molecular torch, or amolecular beacon.
 11. The method of claim 5, wherein the HPV33 detectionprobe further comprises a non-target-hybridizing sequence.
 12. Themethod of claim 11, wherein the HPV33 detection probe is a hairpindetection probe.
 13. The method of claim 6, wherein the HPV31 detectionprobe comprises a target-hybridizing sequence that is from about 14 toabout 40 nucleotides in length and is configured to specificallyhybridize to a target sequence contained within SEQ ID NO:84 from aboutnucleotide position 675 to about nucleotide position
 735. 14. The methodof claim 13, wherein the HPV31 detection probe target-hybridizingsequence is selected from the group consisting of SEQ ID NOs: 59-65. 15.The method of claim 13, wherein the HPV31 detection probe comprises alabel selected from the group consisting of (a) a chemiluminescentlabel; (b) a fluorescent label; (c) a quencher; and (d) a combination oftwo or more of (a), (b), and (c).
 16. The method of claim 6, wherein thedetection step (d′) occurs during the amplification step (c′).
 17. Themethod of claim 16, wherein the HPV31 detection probe is a TaqMandetection probe, molecular torch, or a molecular beacon.
 18. The methodof claim 13, wherein the HPV31 detection probe further comprises anon-target-hybridizing sequence.
 19. The method of claim 18, wherein theHPV31 detection probe is a hairpin detection probe.
 20. The method ofclaim 6, wherein the detection step (d) comprises contacting theamplification reaction of step (c) with an HPV33 detection probeoligomer configured to specifically hybridize to the HPV33 amplificationproduct under conditions whereby the presence or absence of the HPV33amplification product is determined, thereby indicating the presence orabsence of HPV33 in said sample; wherein the detection step (d′)comprises contacting the amplification reaction of step (c′) with anHPV31 detection probe oligomer configured to specifically hybridize tothe HPV31 amplification product under conditions whereby the presence orabsence of the HPV31 amplification product is determined, therebyindicating the presence or absence of HPV31 in said sample; wherein step(c) is performed simultaneously with step (c′) in the same amplificationreaction mixture and step (d) is performed simultaneously with step (d′)in the same detection reaction mixture; and wherein the HPV33 and HPV31detection probe oligomers are differentially labeled.
 21. The method ofclaim 20, wherein each of the HPV33 and HPV31 detection probe oligomerscomprises a label independently selected from the group consisting of(a) a chemiluminescent label and (b) a fluorescent label.
 22. The methodof claim 21, wherein the chemiluminescent labels for the HPV33 and HPV31detection probe oligomers are characterized by different light emissionkinetics sufficient to distinguish between HPV33-specific andHPV31-specific chemiluminescent signals.